CN101558168A - Detection of bacterium by utilizing DnaJ gene and use thereof - Google Patents

Abstract

The invention discloses a method for detecting bacterial strains in a simple manner. Specifically, the present invention discloses a method for detecting bacteria species, which can detect bacteria selected from Staphylococcus (Staphylococcus) bacteria, Streptococcus (Streptococcus) bacteria, Klebsiella (Klebsiella) bacteria, Escherichia, Mycobacterium, Legionella, Vibrio, Bacillus, Neisseria ) bacteria, Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Listeria, Salmonella and at least one bacterial strain in the genus Yersinia (Yersinia) bacterium, and the method comprises the following steps: (a) making the test sample which may contain the nucleic acid of the bacterial strain to be detected and the bacterial strain to be detected contacting a nucleic acid molecule to which at least a portion of the DnaJ gene of the strain hybridizes; and (b) detecting the presence or absence of hybridization between said nucleic acid molecule and a nucleic acid contained in said test sample.

Description

Utilize the Bacteria Detection and the application thereof of DnaJ gene

Technical field

The present invention relates to utilize the Bacteria Detection and the utilization thereof of DnaJ gene, in detail, relate to and utilize the DnaJ gene to come the method for bacterial detection, reach the nucleic acid molecule that uses in this method, and this method and the application of this nucleic acid molecule in diagnosis and evaluation.

Background technology

All the time, detection of various bacteriums such as pathogenetic bacteria and evaluation need to use special reagent and masterful technique, and use special reagent and masterful technique to carry out the cultivation of target bacteria.Growth of pathogenic bacteria is attended by danger to a certain degree, in addition, when imagination has the pathogenic bacteria of multiple possibility, need cultivate under various different conditions.And,, also need to investigate in minute detail for example biochemical characteristic in order to identify bacteria culture.Therefore, the detection of detection of bacterium and evaluation, particularly pathogenetic bacteria and evaluation not only have some danger, and need expensive energy and time.

On the other hand, as the method for classification and the evaluation of bacterium, brought into use the base sequence of 16SrDNA.If utilize the base sequence of such gene, then can expect to detect in the short period of time and identify bacterium and bacteria culture.

But, in the base sequence accumulation of 16S rDNA, when the base sequence of 16S rDNA has at least 98% similarity between the bacteria culture of independent and nearly edge, often can not discern bacterial classification (non-patent literature 1).Therefore, if do not implement the method for the similarity level of more complicated mensuration whole chromosome gene, then can not realize determine (non-patent literature 2) of bacterial classification.For this reason, need the data accumulation of the sequence of variation higher level, ( non-patent literature 3,4 and 5) carried out in the accumulation of the isogenic base sequence data of GyrB or ropB.

A kind of such house-keeping gene is heat shock protein(HSP) DnaJ.It is reported that the DnaJ gene of encoding D naJ can be used for the bacterial classification of Mycobacterium (Mycobacterium) bacterium classify (non-patent literature 6).In addition, it is reported, DnaJ gene pleiomorphism general (non-patent literature 7) in the serotype of mycobacterium avium (Mycobacterium avium) though and only be the short sequence that 200 bases are arranged, but the DnaJ gene is very conservative between serotype, the difference of sequence big (non-patent literature 8) in the bacterial classification level of same genus.In addition, it is reported also that in legionella (Legionella) belonged to, the DnaJ gene was conservative between the serotype of legionella pneumophilia (L.pneumophila), but at the horizontal polymorphism level of bacterial classification higher (non-patent literature 9).

On the other hand, catch, require to detect rapidly and reliably and identify pathogenic micro-organism in the sample of from food or animal, gathering consumingly in prevention or treatment.Be to identify pathogenic micro-organism, use respectively with multiple pathogenic micro-organism in the corresponding multiple primer of a kind of characteristic target nucleic acid right, the multiplex PCR method of implementing PCR is useful.In multiplex PCR, in a pipe, use multiple primer to make the target nucleic acid amplification.For such multiplex PCR method, carrying out various investigations (patent documentation 1～4) at present.Multiplex PCR pathogenic micro-organism such as identifying virus and bacterium in treatment is infected is of great use.In order to identify pathogenic micro-organism, need the specific sequence in the genome of target pathogenic micro-organism.

Non-patent literature 1:Fox, G.E.et al.1992.Int.J.Syst.Bacteriol.42:166-170

Non-patent literature 2:Ezaki, T.et al.1989.Int.J.Syst.Bacteriol.Amano, M.et al.2005.Microbiol.Immunol.49:255-263

Non-patent literature 3:Yamamoto, S.and S.Harayama.1995.Appl.Environ.Microbiol.61:1104-1109.,

Non-patent literature 4:Yamamoto, S.and S.Harayama.1996.Int.J.Syst.Bacteriol.46:506-511

Non-patent literature 5:Cheunoy, W.et al.2005.Diagn.Microbiol.Infect.Dis.51:165-171

Non-patent literature 6:Takewaki, S.et al.1994.Int.J.Syst.Bacteriol.44:159-166

Non-patent literature 7:Victor, TC.et al.1996.J.Med.Microbiol.44:332-339

Non-patent literature 8:Morita, Y.et al.2004.J.Med.Microbiol.53:813-817

Non-patent literature 9:Liu, H.et al.2003.Microbiol.Immunol.47:859-869

Summary of the invention

Usually, base sequence in a certain gene has specificity with respect to the related microorganisms of other genus, and in the bacterial classification level in same genus specificity is arranged also, and conservative to a certain extent in the bacterial strain level of base sequence in bacterial classification, just can only use this gene test or identify specific bacterial classification.That is, determine that whether special genes can be used to detect and identify specific microbial strains, need check order widely to this specific gene that is included in a plurality of bacterial strains in the specified strain.

But, for the DnaJ gene, only be, and check order, also unexposed sequence information the bacterial strain level in these bacterial classifications in the serotype level for specific bacterial classification.The DnaJ gene does not also carry out aforesaid genetic analysis widely.Therefore, so far, whether not clear DnaJ gene has the specific base sequence that can be used to detect or identify bacterial classification separately.And,, need check order to a large amount of bacterial strains because the existence of the polymorphism between the bacterial strain will be found sequence conservative between the bacterial classification.

In addition, when pathogenic micro-organisms such as evaluation bacterium, if use multiple (2～10 kinds) primer with concentration (about 0.2 μ M～0.8 μ M) identical when using multiple primer respectively, produce then that vying each other between primer, amplification efficiency are reduced, the amplified production amount reduces, thereby causes detecting amplified production.In addition, if reduce primer concentration, then amplification efficiency reduces, and at this moment, can not detect amplified production too.The kind of the primer of practicality that therefore, can disposable combination is essentially about 6 kinds.

In addition, implement multiplex PCR in order to utilize multiple primer, the specificity of primer is very important.In multiplex PCR, need the special sequence of target bacterial classification corresponding to multiple pathogenic micro-organism.But for multiple pathogenic micro-organism, design can be used simultaneously and the primer of the target that the bacterial classification that increases is special is very difficult.

One of purpose of the present invention is, provides and can easily detect or identify the method for bacteria culture, and be used for nucleic acid molecule wherein.In addition, other purpose of the present invention is, the method that detects or identify bacteria culture in high sensitivity is provided, and is used for nucleic acid molecule wherein.In addition, other purpose of the present invention also is, is provided for detecting or identifying the method for bacteria culture more than 2 kinds, and is used for nucleic acid molecule wherein.In addition, one of purpose of the present invention is, the amplification method of the microorganism that utilizes multiplex PCR that is suitable for microorganisms such as rapid bacterial detection is provided, and is used for its test kit etc.

The inventor has carried out the order-checking of DnaJ gene to 680 bacterial classifications and 1250 bacterial strains, by comparing its result, find, sequence similarity between the bacterial classification of 16S rDNA gene is more than 95%, DnaJ gene similarity between bacterial classification in all belong to basically is 75～90%, and, the sequence information of the bacterial strain level in the same bacterial classification, the contriver only finds to extract the special base sequence that just can identify bacterial classification from the DnaJ gene, has finished the present invention based on above-mentioned discovery.That is,, provide following method according to the present invention.

According to the present invention, a kind of method of bacterial classification of bacterial detection is provided, wherein, to being selected from Staphylococcus (Staphylococcus), streptococcus (Streptococcus), klebsiella (Klebsiella), Escherichia (Escherichia), Mycobacterium (Mycobacterium), legionella (Legionella), Vibrio (Vibrio), bacillus (Bacillus), Neisseria (Neisseria), campylobacter (Campylobacter), chlamydiaceae (Chlamydia), have a liking for chlamydiaceae (Chlamydophila), Mycoplasma (Mycoplasma), Listera belongs to (Listeria), the bacteria culture more than a kind or 2 kinds of Salmonellas (Salmonella) and Yersinia (Yersinia) is as detected object, and this method comprises step:

(a) test sample that makes the nucleic acid that may contain the detected object bacteria culture and the step that contacts with the nucleic acid molecule of at least a portion hybridization of the DnaJ gene of detected object bacteria culture,

(b) detect the step whether nucleic acid in described nucleic acid molecule and the described test sample hybridizes.

In the present invention, described (a) step is preferably the step that described nucleic acid samples and the described nucleic acid molecule more than 2 kinds with at least a portion hybridization separately of the DnaJ gene of bacterial classification more than 2 kinds are contacted simultaneously.In addition, described nucleic acid molecule can comprise by the base sequence of the DnaJ gene of the bacterial strain that will belong to same bacterial classification compares a kind or the special base sequence of multiple bacterial classification that extracts, and maybe can comprise identical with this base sequence basically a kind or multiple base sequence.And described bacterium as detected object can be for being selected from streptococcus pyogenes (Streptococcuspyogenes), streptococcus agalactiae (Streptococcus agalactiae), streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus aureus (Staphylococcus aureus), Klebsiella pneumoniae (Klebsiella pneumoniae), intestinal bacteria (Escherichia coli), mycobacterium tuberculosis (Mycobacterium tuberculosis), mycoplasma pneumoniae (Mycoplasmapneumoniae), Mycoplasma genitarium, vibrio cholerae (Vibrio cholerae), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Vibrio vulnificus (Vibrio vulnificus), vibrio fluvialis (Vibrio fluvialis), vibrio alginolyticus (Vibrio alginolyticus), Neisseria meningitidis (Neisseria meningitides), Diplococcus gonorrhoeae (Neisseria gonorrhoae), bacillus cereus (Bacillus cereus), chlamydia trachomatis (Chlamydia trachomatis), pneumonia is had a liking for 1 or multiple bacterial classification in chlamydozoan (Chlamydophila pneumoniae) and the campylobacter jejuni (Campylobacterjejuni).When detecting the bacteria culture of respective specific, can use specific nucleic acid molecule.

Described detected object bacterium can be to be selected from streptococcus (Streptococcus), Staphylococcus (Staphylococcus), klebsiella (Klebsiella), Escherichia (Escherichia), Mycobacterium (Mycobacterium), legionella (Legionella), chlamydiaceae (Chlamydia), have a liking for chlamydiaceae (Chlamydophila), 1 kind or multiple bacterial classification of Neisseria (Neisseria) and Mycoplasma (Mycoplasma).These bacteriums are pathogenic micro-organisms of pneumonia and/or pharyngitis.Described bacterium can be to be selected from streptococcus pyogenes (Streptococcus pyogenes), streptococcus agalactiae (Streptococcus agalactiae), streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus aureus (Staphylococcusaureus), Klebsiella pneumoniae (Klebsiella pneumoniae), intestinal bacteria (Escherichia coli), mycobacterium tuberculosis (Mycobacterium tuberculosis), legionella pneumophilia (Legionella pneumophila), chlamydia trachomatis (Chlamydia trachomatis), pneumonia is had a liking for chlamydozoan (Chlamydophila pneumoniae), Diplococcus gonorrhoeae (Neisseriagonorrhoae), arbitrary bacterial classification in Neisseria meningitidis (Neisseria meningitides) and the mycoplasma pneumoniae (Mycoplasma pneumoniae), at this moment, described nucleic acid molecule can comprise SEQ ID NO:1～42, in SEQ ID NO:131～134 and SEQ ID NO:89～116 a kind or multiple base sequence or with its same basically a kind or multiple base sequence.

Perhaps, described bacterium can be the bacterial classification more than a kind or 2 kinds that is selected from Vibrio (Vibrio), campylobacter (Campylobacter), Staphylococcus (Staphylococcus), bacillus (Bacillus), Escherichia (Escherichia), Listera genus (Listeria), Salmonellas (Salmonella) and Yersinia (Yersinia).These bacterium are pathogenic bacterias of diarrhoea and/or food poisoning.Described bacterium can be for being selected from vibrio cholerae (Vibriocholerae), Vibrio mimicus (Vibrio mimicus), Vibrio parahaemolyticus (Vibrioparahaemolyticus), vibrio alginolyticus (Vibrio alginolyticus), vibrio fluvialis (Vibriofluvialis), Vibrio vulnificus (Vibrio vulnificus), Vibrio vulnificus (Vibrio vulnificus), campylobacter jejuni (Campylobacter jejuni), streptococcus aureus (Staphylococcusaureus), arbitrary bacterial classification in bacillus cereus (Bacillus cereus) and the intestinal bacteria (Escherichia coli), described in this case nucleic acid molecule can comprise SEQ ID NO:43～70, SEQ ID NO:134, SEQ ID NO:87～88, SEQ ID NO:97～98,1 kind or multiple base sequence or a kind or the multiple base sequence substantially the same in SEQ ID NO:101～102 and SEQ ID NO:117～130 with it.When described bacterium is monocyte hyperplasia Li Site bacterium (Listeria monocytogenes), described nucleic acid molecule can comprise base sequence or the base sequence substantially the same with it that is selected from SEQ ID NO:135～136 or SEQ ID NO:137～138, when described bacterium is Salmonella enteritidis (Salmonella enteritidis), described nucleic acid molecule can comprise base sequence or the base sequence substantially the same with it that is selected from SEQ ID NO:139～140, when described bacterium was Yersinia enterocolitica (Yersinia enterocolitica), described nucleic acid molecule can comprise the base sequence or the base sequence substantially the same with it of SEQ ID NO:143～144.

In addition, described bacterium can be for being selected from 1 in Neisseria (Neisseria), chlamydiaceae (Chlamydia) and the Mycoplasma (Mycoplasma) or a plurality of kinds.These bacteriums are pathogenic bacterias of venereal disease.When described bacterium was Diplococcus gonorrhoeae (Neisseria gonorrhoae), Neisseria meningitidis (Neisseria meningitides), chlamydia trachomatis (Chlamydia trachomatis) or Mycoplasma genitarium, described nucleic acid molecule can comprise base sequence more than a kind or 2 kinds or the base sequence substantially the same with it that is selected from SEQ ID NO:71～86, SEQ ID NO:105～106 and SEQ ID NO:109～114.

According to the present invention, a kind of method of identifying bacteria culture also is provided, wherein, to be selected from streptococcus (Streptococcus) bacterium, Staphylococcus (Staphylococcus) bacterium, enterococcus spp (Enterococcus) bacterium, Peptostreptococcus (PeptoStreptococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, enterobacter (Enterobacter) bacterium, legionella (Legionella) bacterium, Neisseria (Neisseria) bacterium, bacillus (Bacillus) bacterium, Helicobacterium (Helicobacter) bacterium, Corynebacterium (Corynebacterium) bacterium, Rhodopseudomonas (Pseudomonas) bacterium, Bai Huoerde Bacillaceae (Burkholderia) bacterium, Haemophilus spp (Haemophilus) bacterium, Bacteroides (Bacteroides) bacterium, enterococcus spp (Enterococcus) bacterium, Nuo Kaer Pseudomonas (Nocardia) bacterium, general Bordetella (Prevotella) bacterium, Neisseria (Neisseria) bacterium, Moraxella (Moraxella) bacterium, Flavobacterium (Flavobacterium) belongs to bacterium, fusobacterium (Clostridium) bacterium, treponema (Treponema) bacterium, Sphingobacterium (Sphingobacterium) bacterium, leptospira (Leptospira) bacterium, campylobacter (Campylobacter) bacterium, Listera belongs to (Listeria) bacterium, bacteria culture more than a kind or 2 kinds in Salmonellas (Salmonella) bacterium and Yersinia (Yersinia) bacterium as identify object to bacteria-like, this method may further comprise the steps:

(a) obtain to contain at least a portion base sequence to the DnaJ gene in the test sample of the nucleic acid of bacteria-like.

This authentication method preferably includes (b) step, and the base sequence of at least a portion base sequence of the DnaJ gene that soon obtains from described test sample and the DnaJ gene of bacterium to be identified contrasts, and identifies the step of the bacterial classification of the bacterium in the described test sample.

According to the present invention, also provide a kind of Bacteria Identification program to be used to identify bacterium, wherein, to be selected from streptococcus (Streptococcus) bacterium, Staphylococcus (Staphylococcus) bacterium, enterococcus spp (Enterococcus) bacterium, Peptostreptococcus (PeptoStreptococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, enterobacter (Enterobacter) bacterium, legionella (Legionella) bacterium, Neisseria (Neisseria) bacterium, bacillus (Bacillus) bacterium, Helicobacterium (Helicobacter) bacterium, Corynebacterium (Corynebacterium) bacterium, Rhodopseudomonas (Pseudomonas) bacterium, Bai Huoerde Bacillaceae (Burkholderia) bacterium, Haemophilus spp (Haemophilus) bacterium, Bacteroides (Bacteroides) bacterium, enterococcus spp (Enterococcus) bacterium, Nuo Kaer Pseudomonas (Nocardia) bacterium, general Bordetella (Prevotella) bacterium, Neisseria (Neisseria) bacterium, Moraxella (Moraxella) bacterium, Flavobacterium (Flavobacterium) belongs to bacterium, fusobacterium (Clostridium) bacterium, treponema (Treponema) bacterium, Sphingobacterium (Sphingobacterium) bacterium, bacteria culture more than a kind or 2 kinds in leptospira (Leptospira) bacterium and campylobacter (Campylobacter) bacterium is as the bacteria culture of identifying object, this program is carried out following steps on 1 or 2 above computer, be about to the base sequence contrast of the DnaJ gene of at least a portion base sequence of the DnaJ gene that from the test sample of the nucleic acid that may contain bacterium to be identified, obtains and bacterium to be identified, identify the step of the bacterial classification of the bacterium in the described test sample.

According to the present invention, a kind of nucleic acid molecule that is used to detect and identify bacterium also is provided, wherein, to be selected from streptococcus (Streptococcus) bacterium, Staphylococcus (Staphylococcus) bacterium, enterococcus spp (Enterococcus) bacterium, Peptostreptococcus (PeptoStreptococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, enterobacter (Enterobacter) bacterium, legionella (Legionella) bacterium, Neisseria (Neisseria) bacterium, bacillus (Bacillus) bacterium, Helicobacterium (Helicobacter) bacterium, Corynebacterium (Corynebacterium) bacterium, Rhodopseudomonas (Pseudomonas) bacterium, Bai Huoerde Bacillaceae (Burkholderia) bacterium, Haemophilus spp (Haemophilus) bacterium, Bacteroides (Bacteroides) bacterium, enterococcus spp (Enterococcus) bacterium, Nuo Kaer Pseudomonas (Nocardia) bacterium, general Bordetella (Prevotella) bacterium, Neisseria (Neisseria) bacterium, Moraxella (Moraxella) bacterium, Flavobacterium (Flavobacterium) bacterium, fusobacterium (Clostridium) bacterium, treponema (Treponema) bacterium, Sphingobacterium (Sphingobacterium) bacterium, leptospira (Leptospira) bacterium, campylobacter (Campylobacter) bacterium, Listera belongs to (Listeria) bacterium, the object of the bacteria culture more than a kind or 2 kinds in Salmonellas (Salmonella) bacterium and Yersinia (Yersinia) bacterium for detecting and identifying, and at least a portion of the DnaJ gene of described nucleic acid molecule and bacterium to be identified hybridization.Described nucleic acid molecule can be primer set for amplification, also can be probe.In addition, be selected from the form that can be immobilized onto the cured body in the solid phase more than a kind or 2 kinds of above nucleic acid molecule for this nucleic acid molecule, also can be for comprising the Bacteria Identification of nucleic acid molecule more than a kind or 2 kinds that is selected from these nucleic acid molecule and the form of detection kit.

In addition, the inventor also finds, is divided into 2 by the function with primer, can be rapidly and detect microorganism in high sensitivity.Promptly, the inventor finds, by the 1st primer sets and the 2nd primer sets are made up, even under the situation that a plurality of primer sets that make at multiple target act on simultaneously, also sensitivity fully detects target sequence, wherein, the 1st primer sets has sequence and the unannealed flag sequence to target nucleic acid that specificity is annealed to target nucleic acid, and the 2nd primer sets has the flag sequence substantially the same with the flag sequence of the 1st primer sets.In addition, the inventor also finds, for produce the primer mixture that can detect multiple microorganism (below be also referred to as mix primer (Cocktail primer), DnaJ gene by each microbial species of target, thereby can easily design the bacterial classification Auele Specific Primer can single basically PCR reaction cycle amplification efficiency amplified target and sensitivity detecting well well, finally finish the present invention thus.According to the present invention, also provide following method.

In addition, according to the present invention, the method of at least a target nucleic acid that increases is provided, wherein, may further comprise the steps, that is, use to have flag sequence and described target nucleic acid is had the 1st primer sets and at least a kind of the 2nd primer sets with flag sequence substantially the same with the flag sequence of described the 1st primer sets of specific base sequence, implement the step of polymerase chain reaction.

Described polymerase chain reaction step can be the step of the amplified production implementing to utilize described the 1st primer sets to increase described target nucleic acid and utilize described the 2nd primer sets amplification to utilize described the 1st primer sets to increase to obtain.And described polymerase chain reaction step can be for implementing the step of single basically PCR reaction cycle.

Described polymerase chain reaction step can be for implementing the step of single PCR reaction cycle, wherein, the primer concentration of described the 1st primer sets is below the above 0.1 μ M of 0.005 μ M, the primer concentration of described the 2nd primer sets is more than 10 times below 50 times of primer concentration of described the 1st primer sets, utilize described the 2nd primer sets to increase and obtain amplified production be the following nucleic acid of 250 bases.

According to the present invention, a kind of method based on multiplex PCR diagnosis pathogenic micro-organism also is provided, described pathogenic micro-organism is for being selected from Staphylococcus (Staphylococcus) bacterium, streptococcus (Streptococcus) bacterium, Serratia (Serratia) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, Mycobacterium (Mycobacterium) bacterium, legionella (Legionella) bacterium, Vibrio (Vibrio) bacterium, bacillus (Bacillus) bacterium, Neisseria (Neisseria) bacterium, campylobacter (Campylobacter) bacterium, chlamydiaceae (Chlamydia) bacterium, have a liking for chlamydiaceae (Chlamydophila) bacterium, Mycoplasma (Mycoplasma) bacterium, Listera belongs to (Listeria) bacterium, bacterial classification more than a kind or 2 kinds in Salmonellas (Salmonella) bacterium and Yersinia (Yersinia) bacterium, this method comprises uses at least a the 1st primer sets and at least a the 2nd primer sets, to containing the step of implementing polymerase chain reaction from the test sample of the nucleic acid of described pathogenic micro-organism, contain the step of the amplified production of described target nucleic acid with detection, described the 1st primer sets has flag sequence and selectively annealed base sequence to the target nucleic acid (preferably on the DnaJ gene) that described pathogenic micro-organism had, and described the 2nd primer sets has the flag sequence substantially the same with the flag sequence of described the 1st primer sets.In the method, described polymerase chain reaction step is preferably the step of implementing single PCR reaction cycle.

According to the present invention, a kind of test kit also is provided, it is the test kit that comprises the primer sets that is used for the target nucleic acid by at least a kind of microorganism of multiplex PCR amplification, wherein, described test kit comprises the 1st primer sets and the 2nd primer sets, described the 1st primer sets has flag sequence and selectively annealed base sequence to the target nucleic acid (preferably on the DnaJ gene) that described pathogenic micro-organism had, and described the 2nd primer sets has the flag sequence substantially the same with the flag sequence of described the 1st primer sets.

Description of drawings

Figure 1A is the figure of comparison 16S rDNA sequence.Between two bacteria cultures, have only 12 bases (1.2%) that difference is arranged.

Figure 1B is the figure that shows the polymorphism of DnaJ sequence.Between two bacteria cultures, 183 bases (20.6%) have difference.

Fig. 2 is the figure that shows the DnaJ primer sequence of streptococcus aureus (S.aureus).

Fig. 3 shows the figure utilize the result that independent primer (primer solution B) and multi-primers (primer solution A) increase.

Fig. 4 is the figure that strengthens the result who is presented at the DNA acquisition of increasing in Light Cycler (Roche Holding Ag) kapillary by monitoring CYBR green fluorescence intensity.

Fig. 5 shows the figure that confirms the result of pcr amplification product with agar electrophoresis.

Fig. 6 is the figure that relatively utilizes the detection sensitivity of gene amplification acquisition with electrophoretic method.

Fig. 7 strengthens by monitoring CYBR green fluorescence intensity, is presented at the result's of the DNA that increases in the kapillary of Light Cycler (Roche Holding Ag) figure.

Fig. 8 has shown 55 bacterial classifications of Mycobacterium (Mycobacterium) of DnaJ gene sequencing and the type strain of 3 subspecies.

Fig. 9 is the figure of the sequence of comparison DnaJ gene and RpoB gene.

Figure 10 is the figure of the base sequence of comparison range gene.

Figure 11 is the figure of similarity of the base sequence of the base sequence of comparison 16S rDNA gene and DnaJ gene.

Figure 12 has shown and has utilized result that the various bacterial strains of DnaJ gene pairs identify and the result who utilizes biochemical characteristic that various bacterial strains are identified.

Figure 13 has shown the result who the 16 strain clinical separation strains (the KPM strain among the figure) that are referred to as mycobacterium avium-mycobacterium intracellulare (M.avium-M.intracellulare) group (MAC group) is identified based on the base sequence of DnaJ gene.

Figure 14 shows the 1st primer sets used in the amplification method of the present invention and the figure of the relation between the 2nd primer sets and the target nucleic acid.

Figure 15 has shown and has been designed for the primer sequence of legionella pneumophilia (Legionella pneumophila) GTC1279DNA as target nucleic acid.

Figure 16 is the figure that shows the amplification monitoring result in the PCR step of implementing among the embodiment 8.

Figure 17 is the figure that shows the amplification monitoring result in the PCR step of implementing among the embodiment 9.

Figure 18 has shown the primer sequence of the bacterial pathogen that is used to detect diarrhoea.

Figure 19 has shown the primer sequence of the viral pathogens that is used to detect diarrhoea.

Figure 20 has shown the probe sequence that is immobilized among the embodiment 10 on the silicon substrate.

Fluorescence when Figure 21 has shown the probe hybridization of PCR product among the embodiment 10 and silicon substrate.

Figure 22 has shown the primer sequence that is used to detect the STD pathogenic micro-organism.

Figure 23 has shown the probe sequence of the amplified production in the PCR product that is used for detecting embodiment 11.

Figure 24 has shown the fluorescence intensity of the hybridization product that obtains among the embodiment 11.

Figure 25 has shown the primer that constitutes 4 kinds of mix primer of preparation among the embodiment 12.

Figure 26 has shown as the CTm value of carrying out the result of PCR in embodiment 12.

Embodiment

Detection method disclosed by the invention, by making described test sample and contact with the nucleic acid molecule of at least a portion hybridization of the DnaJ gene of bacterial classification to be detected, whether detection exists hybridization, can detect specific microbial strains thus.Though DnaJ gene similarity between microbial strains is lower, between the bacterial strain of same bacterial classification, guard at certain level or more than the certain level.Therefore, can easily extract the special base sequence of bacterial classification, and easily detect specific bacterial classification by using this base sequence.Particularly, therefore,, also can sensitivity detect specified strain well even be between sibling species because the similarity between bacterial classification is low.And,, therefore, can design special primer etc. because similarity is lower between bacterial classification.Its result even use the mixture of a plurality of primer sets, also can detect specific bacterial classification.Therefore, can detect a plurality of bacterial classifications with the a kind of primer mixture that contains multiple primer sets.Here, because being earn a bare living indispensable house-keeping gene and all microbial strains, the DnaJ gene all has the DnaJ gene, so detection of DnaJ gene pairs microbial strains and evaluation are favourable.

In addition, authentication method of the present invention, in view of the similarity of DnaJ gene between bacteria culture low, therefore, base sequence by for example measuring the DnaJ gene in the bacterium to be identified or DnaJ gene is analyzed with specified probe hybridization, compare by analytical results then, can detect microbial strains to be identified with the base sequence of the DnaJ gene of existing nearly edge bacterial classification.

Below to embodiments of the present invention, be described in detail at nucleic acid molecule that uses in for example method of detecting bacterium of the present invention and authentication method and these methods etc.

(detection method of bacterium)

(bacterium to be detected)

Method of detecting bacterium target of the present invention is selected from Staphylococcus (Staphylococcus) bacterium, streptococcus (Streptococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, Mycobacterium (Mycobacterium) bacterium, legionella (Legionella) bacterium, Vibrio (Vibrio) bacterium, bacillus (Bacillus) bacterium, Neisseria (Neisseria) bacterium, campylobacter (Campylobacter) bacterium, chlamydiaceae (Chlamydia) bacterium, have a liking for chlamydiaceae (Chlamydophila) bacterium, Mycoplasma (Mycoplasma) bacterium, Listera belongs to (Listeria) bacterium, the bacterium bacterium bacterial classification more than a kind or 2 kinds in Salmonellas (Salmonella) bacterium and Yersinia (Yersinia) bacterium is the detection of the above bacterium bacterium bacterial classification of detected object kind.In belonging to the bacteria culture of these genus, because the similarity of the base sequence of DnaJ gene is lower than the similarity of the base sequence of 16S rDNA gene, find conservative bacterial classification specificity base sequence between the bacterial strain in same bacterial classification easily, therefore make the DnaJ gene be suitable for using the DnaJ gene to be applicable to the bacterial detection bacterial classification.For example, for the DnaJ gene, in Staphylococcus (Staphylococcus) bacterium, the base sequence similarity average out to 97.4% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 77.6% of DnaJ gene between bacterial classification, in streptococcus (Streptococcus) bacterium, the base sequence similarity average out to 95.8% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 76.4% of DnaJ gene between bacterial classification, in Mycobacterium (Mycobacterium) bacterium, the base sequence similarity average out to 93% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 83.7% of DnaJ gene between bacterial classification, in legionella (Legionella) bacterium, the similarity average out to 96.1% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 82.5% of DnaJ gene between bacterial classification.For Vibrio (Vibrio) bacterium, the base sequence similarity average out to 97.4% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 82% of DnaJ gene between bacterial classification, for Aeromonas (Aeromonas) bacterium, the base sequence similarity average out to 98.8% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 89.8% of DnaJ gene between bacterial classification, for enterobacteriaceae (Family Enterobacteriaceae) bacterium, the base sequence similarity average out to 97.1% of 16S rDNA gene between bacterial classification, the base sequence similarity average out to 87.6% of DnaJ gene between bacterial classification.

Wherein, bacterial classification to be detected is preferably klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, Vibrio (Vibrio) bacterium, bacillus (Bacillus) bacterium, Neisseria (Neisseria) bacterium, campylobacter (Campylobacter) bacterium, chlamydiaceae (Chlamydia) bacterium, have a liking for chlamydiaceae (Chlamydophila) bacterium, Mycoplasma (Mycoplasma) bacterium, Listera belongs to (Listeria) bacterium, the bacterial classification of Salmonellas (Salmonella) bacterium and Yersinia (Yersinia) bacterium.This be because, utilize genetic engineering technique to be difficult to detect and identify the bacteria culture of these genus, but utilize the DnaJ gene, can detect by the method for genetic engineering and identify.

Even in the higher enterobacteriaceae (Enterobacteriaceae) of 16S rDNA gene similarity, when detecting difference between the bacterial classification in single genus, in 4 bacterial classifications of klebsiella (Klebsiella), only there is 1.4% difference in 16S rDNA gene, and there is 7.8% difference in the DnaJ gene.In Serratia (Serratia), there is 2.3% difference in 16S rDNA gene, and there is 5.5% difference in the DnaJ gene.In salmonella (Salmonella), there is 1.4% difference in 16S rDNA gene, and there is 6.3% difference in the DnaJ gene, shows higher polymorphism.Thus, by using the DnaJ gene, bacterial detection and identify bacterial classification more suitably then.

In detection method of the present invention, by using the mixture of the nucleic acid molecule detect multiple microbial strains respectively, the bacterial classification that can also target is used to detect more than 2 kinds.This be because, since the base sequence of DnaJ gene between belonging to and the similarity between microbial strains low, between bacterial strain, have the higher sequence site of similarity level, therefore, can design the nucleic acid molecule of mutually noninterfere ground hybridization DnaJ gene.

(test sample)

Detection method of the present invention can may further comprise the steps: the test sample that (a) makes the nucleic acid that may contain bacterium to be detected with contact with nucleic acid molecule as DnaJ gene at least a portion hybridization of the bacteria culture of detected object.Here, the nucleic acid that test sample may contain bacteria culture to be detected gets final product, and is not particularly limited.The illustrative examples of test sample comprises: movement, phlegm, well water, food etc. such as blood, serum, the cell or tissue of collecting from the animal that comprises the people, ight soil and urine.Test sample can be by extract the nucleic acid extraction sample that nucleic acid obtains from the material of such collection, perhaps can be the sample of the amplified production that comprises DnaJ gene or its part (comprising zone to be detected).

(nucleic acid molecule)

The nucleic acid molecule that can be used for the inventive method can comprise the bacteria culture specificity base sequence more than a kind or 2 kinds that the base sequence of the DnaJ gene of the bacterial strain by will belonging to same bacterial classification is compared and extracted.Such nucleic acid molecule is that bacterial classification is special, and can make that the bacterial strain that belongs to same bacteria culture is detected reliably.For the such comparison and the extraction of characteristic sequence, can utilize such as DNASIS pro version (Hitachi's software), Beacon designer 5.1 versions (Molecular Beacon company) and Clustal W softwares such as (free softwares).

Nucleic acid molecule can comprise the bacterial classification specificity base sequence that 1 extraction obtains, and also can comprise more than 2.Perhaps, nucleic acid molecule can comprise and as above operate the substantially the same base sequence of bacterial classification specificity base sequence that extraction obtains.Here, so-called essentially identical sequence can list: based on above-mentioned base sequence and can keep with the specific scope of the hybridization of DnaJ gene in the base sequence modified.Exemplary example comprises above-mentioned base sequence, complementary base sequence with it, the base in above-mentioned base sequence more than 1 or 2 be selected from displacement, insertion, disappearance and add in the mode more than a kind or 2 kinds modify and the base sequence that obtains.In addition, such modification also can be carried out corresponding to the base sequence of the base sequence of the DnaJ gene of specific bacterial strain.

Nucleic acid molecule particularly is primer and the probe that has such specificity base sequence or comprise such base sequence.As long as nucleic acid molecule can carry out just being not particularly limited based on the hybridization of base sequence.Though nucleic acid molecule is DNA in typical case, also can adopt DNA, RNA, DNA-RNA mosaic, the artificial nucleic acid that contains modified base or peptide nucleic acid(PNA) (PNA).Such nucleic acid molecule can also be used multiple fluorochrome, and fluorochrome quencher etc. is modified.

Provided the example of the preferred nucleic acid molecule of each bacteria culture below.When bacterium was streptococcus pyogenes (Streptococcus pyogenes), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ IDNO:1～4 (primer) and SEQ ID NO:89～90 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When described bacteria culture was streptococcus agalactiae (Streptococcus agalactiae), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:5～8 (primers) and SEQ ID NO:91～92 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was streptococcus pneumoniae (Streptococcus pneumoniae), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:9～12 (primers) and SEQ ID NO:93～94 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was streptococcus aureus (Staphylococcus aureus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:13～16 (primers) and SEQ ID NO:97～98 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Klebsiella pneumoniae (Klebsiellapneumoniae), the Exemplary core acid molecule comprised the nucleic acid molecule more than a kind or 2 kinds that contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:17～20 (primers) and SEQ ID NO:109～110 (primers) or the base sequence substantially the same with it.When bacterium was intestinal bacteria (Escherichia coli), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:21～24 (primers) and SEQ ID NO:101～102 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was mycobacterium tuberculosis (Mycobacterium tuberculosis), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:25～28 (primers) and SEQ ID NO:103～104 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.Bacterium be mycobacterium (Mycobacterium spp.) (wherein, except that tuberculosis mycobacterium (M.tuberculosis) group), the Exemplary core acid molecule comprises and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:29～30 (primers) and SEQ ID NO:131～133 (primers) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was legionella pneumophilia (Legionella pneumophila), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:35～38 (primers) and SEQ ID NO:115～116 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was vibrio cholerae (Vibrio cholerae), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:43～46 (primers) and SEQ ID NO:127～128 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Vibrio mimicus (Vibrio mimicus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:43, SEQ ID NO:47～49 (primers) and SEQ ID NO:125～126 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Vibrio parahaemolyticus (Vibrioparahaemolyticus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:43, SEQ ID NO:50～52 (primers) and SEQ ID NO:123～124 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Vibrio vulnificus (Vibrio vulnificus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:43, SEQ ID NO:53～55 (primers) and SEQ ID NO:119～120 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was vibrio fluvialis (Vibrio fluvialis), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:43, SEQ ID NO:134, SEQ ID NO:56～57 (primers) and SEQ ID NO:117～118 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was vibrio alginolyticus (Vibrio alginolyticus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQID NO:43, SEQ ID NO:58～60 (primers) and SEQ ID NO:121～122 (primers) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was bacillus cereus (Bacillus cereus), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:61～66 (primers) and SEQ ID NO:87～88 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was campylobacter jejuni (Campylobacter jejuni), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:67～70 (primers) and SEQ ID NO:129～130 (primers) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Neisseria meningitidis (Neisseriameningitidis), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:83～86 (primers) and SEQ ID NO:111～112 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Diplococcus gonorrhoeae (Neisseria gonorrhoeae), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:79～82 (primers) and SEQ ID NO:113～114 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was chlamydia trachomatis (Chlamydia trachomatis), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:75～78 (primers) and SEQ ID NO:109～110 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.Bacterium is a pneumonia when having a liking for chlamydozoan (Chlamydophila pneumoniae), and the Exemplary core acid molecule comprises and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:39～42 (primers) and SEQ ID NO:107～108 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was Mycoplasma genitarium, the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:71～74 (primers) and SEQ ID NO:105～106 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.When bacterium was mycoplasma pneumoniae (Mycoplasma pneumoniae), the Exemplary core acid molecule comprised and contains the base sequence more than a kind or 2 kinds that is selected from SEQ ID NO:31～34 (primers) and SEQ ID NO:95～96 (probes) or substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.

When detecting the pathogenic bacteria of pneumonia or pharyngitis, organism to be detected can be for from streptococcus (Streptococcus) bacterium, Staphylococcus (Staphylococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, Mycobacterium (Mycobacterium) bacterium, legionella (Legionella) bacterium, chlamydiaceae (Chlamydia) bacterium, have a liking for chlamydiaceae (Chlamydophila) bacterium, bacterium more than a kind or 2 kinds in Neisseria (Neisseria) bacterium and Mycoplasma (Mycoplasma) bacterium.At this moment, bacterium is selected from streptococcus pyogenes (Streptococcus pyogenes), streptococcus agalactiae (Streptococcus agalactiae), streptococcus pneumoniae (Streptococcus pneumoniae), streptococcus aureus (Staphylococcusaureus), Klebsiella pneumoniae (Klebsiella pneumoniae), intestinal bacteria (Escherichia coli), mycobacterium tuberculosis (Mycobacterium tuberculosis), legionella pneumophilia (Legionella pneumophila), chlamydia trachomatis (Chlamydia trachomatis), pneumonia is had a liking for chlamydozoan (Chlamydophila pneumoniae), Diplococcus gonorrhoeae (Neisseriagonorrhoae), when Neisseria meningitidis (Neisseria meningitides) and mycoplasma pneumoniae (Mycoplasma pneumoniae), the Exemplary core acid molecule comprises containing and is selected from SEQ IDNO:1～42, base sequence more than a kind or 2 kinds in SEQ ID NO:131～133 and SEQ ID NO:89～116 (probes) or contain substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.Wherein, preferably will be corresponding to the primer sets combination of each DnaJ gene of bacteria culture more than 2 kinds or more than 3 kinds.

In addition, when detecting the pathogenic bacteria of diarrhoea or food poisoning, organism to be detected can be to belong to the bacterium more than a kind or 2 kinds in (Listeria) bacterium, salmonella (Salmonella) bacterium and Yersinia (Yersinia) bacterium from Vibrio (Vibrio) bacterium, campylobacter (Campylobacter) bacterium, Staphylococcus (Staphylococcus) bacterium, bacillus (Bacillus) bacterium, Escherichia (Escherichia) bacterium, Listera.At this moment, bacterium is for being selected from vibrio cholerae (Vibrio cholerae), Vibrio mimicus (Vibrio mimicus), Vibrio parahaemolyticus (Vibrioparahaemolyticus), vibrio alginolyticus (Vibrio alginolyticus), vibrio fluvialis (Vibriofluvialis), Vibrio vulnificus (Vibrio vulnificus), Vibrio vulnificus (Vibrio vulnificus), campylobacter jejuni (Campylobacter jejuni), streptococcus aureus (Staphylococcusaureus), during a kind of in bacillus cereus (Bacillus cereus) and the intestinal bacteria (Escherichia coli), the Exemplary core acid molecule comprises containing and is selected from SEQ ID NO:43～70, SEQ IDNO:134, SEQ ID NO:87～88, SEQ ID NO:97～98, base sequence more than a kind or 2 kinds in SEQ ID NO:101～102 and SEQ ID NO:117～130 or contain substantially the same with it nucleic acid molecule of base sequence more than a kind or 2 kinds.In addition, when bacterium is monocyte hyperplasia Li Site bacterium (Listeria monocytogenes), the Exemplary core acid molecule can comprise contain SEQ ID NO:135 and 136 or SEQ ID NO:137 and 138 in base sequence or contain the nucleic acid molecule of the base sequence substantially the same with it, when described bacterium is Salmonella enteritidis (Salmonella enteritidis), the Exemplary core acid molecule comprises the nucleic acid molecule that contains base sequence in SEQ ID NO:139 and 140 or contain the base sequence substantially the same with it, when described bacterium was Yersinia enterocolitica (Yersinia enterocolitica), the Exemplary core acid molecule comprised the nucleic acid molecule that contains base sequence in SEQ ID NO:143 and 144 or contain the base sequence substantially the same with it.Wherein, preferably will be corresponding to the primer sets combination of each DnaJ gene of 2 kinds or 3 kinds or above bacteria culture.

When detecting the pathogenic bacteria of venereal disease, organism to be detected can be from the bacterium more than a kind or 2 kinds in Neisseria (Neisseria) bacterium, chlamydiaceae (Chlamydia) bacterium or Mycoplasma (Mycoplasma) bacterium.In this case, when described bacterium was Diplococcus gonorrhoeae (Neisseria gonorrhoae), Neisseria meningitidis (Neisseria meningitides), chlamydia trachomatis (Chlamydia trachomatis) and Mycoplasma genitarium, the Exemplary core acid molecule comprised and contains the nucleic acid molecule that is selected from the base sequence more than a kind or 2 kinds in SEQ ID NO:71～86, SEQ ID NO:105～106 and SEQ ID NO:109～114 or contains the base sequence substantially the same with it.Wherein, preferably will be corresponding to the primer sets combination of each DnaJ gene of bacteria culture more than 2 kinds or 3 kinds.

To be applicable to that preferably the base sequence of nucleic acid molecule and the bacterium name of bacterium to be detected together are recited in following.In addition, put down in writing nucleic acid molecule as primer substantially according to the primer sets of forming by the order of forward primer and reverse primer.

(being preferably used as the base sequence of the nucleic acid molecule of primer)

(primer of the pathogenic bacteria of respiratory tract infection)

Streptococcus pyogenes (Streptococcus pyogenes)

SEQ ID NO：01：5-ATAAGGATGTTCAAGAAGCTTACG

SEQ ID NO：02：5-CACCAAAGCCGCCTTGAG

SEQ ID NO：03：5-TTTTGAAGAGGCTGTATTTGG

SEQ ID NO：04：5-GAGC GGTACCAGGTTTTG

Streptococcus agalactiae (Streptococcus agalactiae)

SEQ ID NO：05：5-GCTTATGAAACCTTGAGTGATAC

SEQ ID NO：06：5-AAACCGCCACCATCGAAG

SEQ ID NO：07：5-GGAGATGACCTACAATATCG

SEQ ID NO：08：5-GGCTAGTACCTGGCTTAG

Streptococcus pneumoniae (Streptococcus pneumoniae)

SEQ ID NO：09：5-GCCTATGAGACTTTGAGTGAC

SEQ ID NO：10：5-ACCAGCTCCACCAAAACC

SEQ ID NO：11：5-GCAATCCAAACGCTCCTC

SEQ ID NO：12：5-TGTACGACAGCCAGCTTC

Streptococcus aureus (Staphylococcus aureus)

SEQ ID NO：13：5-GGACAAGGATTCAATGGCTCT

SEQ ID NO：14：5-TTGCGGTGCATTTGGATCTCT

SEQ ID NO：15：5-GAAGCGGTATTTGGTACAAC

SEQ ID NO：16：5-GCCATTACAGTAACTACAAGTC

Klebsiella pneumoniae (Klebsiella pneumoniae)

SEQ ID NO：17：5-CCCTGTCGGTTAAAATTCC

SEQ ID NO：18：5-CTCAAAGATAGCGTGCTG

SEQ ID NO：19：5-GCGTAGAAAAGACCAAAACC

SEQ ID NO：20：5-CAGGTTCAGGTGAAGCAG

Intestinal bacteria (Escherichia coli)

SEQ ID NO：21：5-TGTTGAGCGCAGCAAAAC

SEQ ID NO：22：5-CAAGACGGATGCGGTCTC

SEQ ID NO：23：5-CTCGAAGAAGCTGTACGTG

SEQ ID NO：24：5-CTGTGTACCTGGTTTTGC

Mycobacterium tuberculosis (Mycobacterium tuberculosis)

SEQ ID NO：25：5-GTTCGACAGCGGCTTTGG

SEQ ID NO：26：5-GAACAAGTCGTTGAGGTTGAAC

SEQ ID NO：27：5-AAGACTTCTACCAGGAGCTG

SEQ ID NO：28：5-ACTTCCGATAGGCACGTTT

Mycobacterium (Mycobacterium spp.) (except that tuberculosis mycobacterium (M.tuberculosis))

SEQ ID NO：29：5-CGIGARTGGGTYGARAARG

SEQ ID NO：30：5-GGIGAYYTITTCGGIGGIYT

SEQ ID NO：131：5-GTGARTGGGTCGARAARGACT

SEQ ID NO：132：5-CGIGTYTCGTCGTAYTCCTT

SEQ ID NO：133：5-CAGCGRTTACCYGCCCA

Mycoplasma pneumoniae (Mycoplasma pneumoniae)

SEQ ID NO：31：5-AGCTTGCTATGCAGTACC

SEQ ID NO：32：5-CTCTTTAAACTTCTCTTCGTTTTG

SEQ ID NO：33：5-TTGAGATGACTAATGGTTGTACTC

SEQ ID NO：34：5-CCCTTCAGCCCCAAAACC

Legionella pneumophilia (Legionella pneumophila)

SEQ ID NO：35：5-ACTGTTTGTGAGGGATCG

SEQ ID NO：36：5-AAAACCTTGTTGAATTCTGAC

SEQ ID NO：37：5-TTGAAGAAGCGGCTATAGG

SEQ ID NO：38：5-CTCACAAACAGTACAAGTACC

Pneumonia is had a liking for chlamydozoan (Chlamydophila pneumoniae)

SEQ ID NO：39：5-TCTCAGTGATCCTCAGAA

SEQ ID NO：40：5-CCAAAGGCTCCCATGAAAG

SEQ ID NO：41：5-TTTGGAATGCGCTCAGATC

SEQ ID NO：42：5-GCTTCTTCAAAAGTCAAATTAATATG

(causing the primer of the microorganism of diarrhoea or food poisoning)

Vibrio cholerae (Vibrio cholerae)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：44；5-AGCAGCTTATGACCAATACGCC

SEQ ID NO：45：5-AAACACGTCACCGAAAATATC

SEQ ID NO：46：5-TTATGACCAATACGGCCATG

Vibrio mimicus (Vibrio mimicus)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：47：5-YCTTGAAGAAGCGGTTCGTGCA

SEQ ID NO：48：5-ATATCGCCAAAGTCAG

SEQ ID NO：49：5-GCATCGGTCAGAATTTCATAC

Vibrio parahaemolyticus (Vibrio parahaemolyticus)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：50：5-TGCGAAGAAAGGCTCATCAGAG

SEQ ID NO：51：5-CCAAAGATGTCGCCGAAG

SEQ ID NO：52：5-CGAAGTTCTAACCGACTCTC

Vibrio vulnificus (Vibrio vulnificus)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：53：5-GTACGAAATTCTGACCGATCAA

SEQ ID NO：54：5-CCACCGCCTTGTTCAAAAG

SEQ ID NO：55：5-ACGAAATTCTGACCGATCC

Vibrio fluvialis (Vibrio fluvialis)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：134：5-GTACGAAATTCTGACCGATCAA

SEQ ID NO：56：5-CAATCTCTTTCGAGCAACCAC

SEQ ID NO：57：5-CAACGTGGTGCGGATCTG

Vibrio alginolyticus (Vibrio alginolyticus)

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：58：5-GATCGAAGTRCCRACACTMGGA

SEQ ID NO：59：5-CCGAATACATCGCCAAAG

SEQ ID NO：60：5-ATGCTGCTTTTGAACAAGG

Bacillus cereus (Bacillus cereus)

SEQ ID NO：61：5-GATCAATTTGGTCATGCTGGT

SEQ ID NO：62：5-GAAGCCACCACCGAAGTC

SEQ ID NO：63：5-AAAGCGTATCGTCGTTTGG

SEQ ID NO：64：5-ATCATCACTTAACACTTCATATGC

SEQ ID NO：65：5-CGCAGTACGATCAATTTGG

SEQ ID NO：66：5-CAAAGAAAGAACTAAATATATCTTC

Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO：67：5-TACTTATAAATGCTCTTGTAAAAC

SEQ ID NO：68：5-CTTTGGACAAGTTTGAAG

SEQ ID NO：69：5-TAGACTTTACTTATAAATGCTCTTG

SEQ ID NO：70：5-GACACTTTGGACAAGTTTG

(primer of the pathogenic bacteria of venereal disease)

Mycoplasma genitarium

SEQ ID NO：71：5-GAAGGGTTAAATGCTTCTGG

SEQ ID NO：72：5-CACCATCCATTCCAAAGG

SEQ ID NO：73：5-AAGGGATTATTATGAAGTTCTAGGG

SEQ ID NO：74：5-CATTTTCTGCTTTATGACGATC

Chlamydia trachomatis (Chlamydophila trachomatis)

SEQ ID NO：75：5-GGGATTTCCTTCTTGATCC

SEQ ID NO：76：5-CATGGGGATGATTTAGTTTTAG

SEQ ID NO：77：5-AAAGAAACCGCCTCCATTA

SEQ ID NO：78：5-GCATGGGGAATATGGAAGAC

Diplococcus gonorrhoeae (Neisseria gonorrhoae)

SEQ ID NO：79：5-GGCGGGGAGTTAGAAGTG

SEQ ID NO：80：5-GACACCCTTACCTTTCACG

SEQ ID NO：81：5-CGGGCAAACATATTAAAGAACC

SEQ ID NO：82：5-CGGGAATATTGACTTCCAC

Neisseria meningitidis (Neisseria meningitidis)

SEQ ID NO：83：5-TTGGAAGTGCCGACCTTG

SEQ ID NO：84：5-GATTTGACACCCTTACCCTTCG

SEQ ID NO：85：5-GGGCAAACACATTAAAGAACC

SEQ ID NO：86：5-GCGGGAATATTGACTTCCA

(being preferably used as the base sequence of the nucleic acid molecule of probe)

Bacillus cereus (Bacillus cereus)

SEQ ID NO：87：TTACCATCCAGACGTAAGTAAAGAAGAA

SEQ ID NO：88：TTAGCCACCGCCGAAGTCTCCTCC

Streptococcus pyogenes (Streptococcus pyogenes)

SEQ ID NO：89：TTCGCCGTATTGATCATAAGCAGCA

SEQ ID NO：90：TTCCTGAGCCTAAACAAGTGCCG

Streptococcus agalactiae (Streptococcus agalactiae)

SEQ ID NO：91：TTAAAACCGCCATTTGCCCCAG

SEQ ID NO：92：TCTTCCTGAACAAGTATGACATGATGAC

Streptococcus pneumoniae (Streptococcus pneumoniae)

SEQ ID NO：93：TTCGTGCTGCCTATGACCAGTATG

SEQ ID NO：94：TCGATGATACTTAACTTCCTTCTCAGT

Mycoplasma pneumoniae (Mycoplasma pneumoniae)

SEQ ID NO：95：TACAGTCTCACCCTCACCCTTATG

SEQ ID NO：96：TTCACAGGCACTACAGGTCACC

Streptococcus aureus (Staphylococcus aureus)

SEQ ID NO：97：TTCAATCCGTAAAGATGTAACATGCG

SEQ ID NO：98：TTCAATCCGTAAAGATGTAACATGCGAA

Klebsiella pneumoniae (Klebsiella pneumoniae)

SEQ ID NO：99：TTCGTACAGATCGCCTGCCG

SEQ ID NO：100：CGCGCCGGCAGGCGATCTGTACGT

Intestinal bacteria (Escherichia coli)

SEQ ID NO：101：AAGAGATCCGCATTCCGACTCTGGAAG

SEQ ID NO：102：TTAAGAGATCCGCATTCCGACT

Mycobacterium tuberculosis (Mycobacterium tuberculosis)

SEQ ID NO：103：TCGGGGTCGGTGGAGACGGCG

SEQ ID NO：104：TTCTCCTCTGATGCCAGTCCTGAAGA

Mycoplasma genitarium

SEQ ID NO：105：TAGCAGGGTTTAATCCTTTTGACATCT

SEQ ID NO：106：TAAACGCTAGTTCTCAAGACATAAA

Pneumonia is had a liking for chlamydozoan (Chlamydophila pneumoniae)

SEQ ID NO：107：TTCGCAAGGCATCTTCCATGTTCC

SEQ ID NO：108：TTTCTTACTGGCTCCTTGACGA

Chlamydia trachomatis (Chlamydia trachomatis)

SEQ ID NO：109：TTAGCCGCATCAACAAATCCAATAGG

SEQ ID NO：110：TTACCGAAATCGCCGCCAAA

Neisseria meningitidis (Neisseria meningitidis)

SEQ ID NO：111：TTCTTGACCGCCTTATTCCGCC

SEQ ID NO：112：TGCCGTGGCGCGGGGCGGAATA

Diplococcus gonorrhoeae (Neisseria gonorrhoeae)

SEQ ID NO：113：TTCTTGACCGCCTTATTCCGCC

SEQ ID NO：114：TGCCGTGGCGTGGGGCGGAATA

Legionella pneumophilia (Legionella pneumophila)

SEQ ID NO：115：TAAGAAGTTGAAATTACCGTTCCAAGAC

SEQ ID NO：116：AAAGAAGTTGAAATTACCGTTCC

Vibrio fluvialis (Vibrio fluvialis)

SEQ ID NO：117：TTCCAGCGTCAGTTCCATGTTG

SEQ ID NO：118：TCGAGCAGCCGCGTACCGCTTCT

Vibrio vulnificus (Vibrio vulnificus)

SEQ ID NO：119：TCCATATTGATCGTAAGCCGCT

SEQ ID NO：120：TCGAGACACCACGAACGGCCTCT

Vibrio alginolyticus (Vibrio alginolyticus)

SEQ ID NO：121：TTAATCAGCACCGCCGCCAC

SEQ ID NO：122：TCGTTACACCACGAACCGCTTCT

Vibrio parahaemolyticus (Vibrio parahaemolyticus)

SEQ ID NO：123：TTCAGCACCGCCTCCACCAA

SEQ ID NO：124：TAGTTACACCACGAACGGCTTCT

Vibrio mimicus (Vibrio mimicus)

SEQ ID NO：125：TTCGAAGCCGCCGCCACCAA

SEQ ID NO：126：TCGAACATCCACGAACCGCTTCT

Vibrio cholerae (Vibrio cholerae)

SEQ ID NO：127：TTAATCAGCACCGCCACCGC

SEQ ID NO：128：TCGAGCAGCCGCGAACCGCTTCT

Campylobacter jejuni (Campylobacter jejuni)

SEQ ID NO：129：TGTAATGGAACAGGGGCTA

SEQ ID NO：130：TCCATCTTTAGCCCCTGTTCCAT

For test sample is contacted with nucleic acid molecule, make DnaJ gene of the microorganism to be detected that may contain in the test sample and nucleic acid molecule should be in the state that to hybridize.For example, DnaJ gene and nucleic acid molecule are coexisted as in the liquid phase, also can make in the liquid phase that one of them is combined in the solid phase, another is included in solid phase contacts.As described later, the step that realizes such contact can be based on the gene amplification method such as PCR, RT-PCR method, transcriptive intermediate TRAP (TMA method), branched DNA method (bDNA method), PCR in real time method, ligase chain reaction method (LCR method), perhaps implements based on the part such as the base sequencing of southern blotting, microarray method, microballon method, the chromatographic nucleic acid hybridization of nucleic acid (DNA).The technology that adopts in this step also can be designed to primer according to for example employed nucleic acid molecule and still be designed to probe and carry out appropriate selection.

The inventive method can comprise the step whether nucleic acid in detection nucleic acid molecule and the test sample hybridizes.Whether such hybridization takes place in order to detect, for example, can carry out by the following method: the nucleic acid that (1) is directly detected in nucleic acid molecule and the test sample has or not specific hybrid, (2) based on having or not specific hybrid between the nucleic acid in nucleic acid molecule and the test sample, detect gene amplification reaction and whether exist, and have or not specific base sequence in (3) detection gene amplification reaction product.

Under the situation of above-mentioned (1), for example, form compound by in nucleic acid molecule and/or the nucleic acid that from test sample, extracts, adding the signal that produces tagged compound that can detection signal or add the variation that produces signal, can detect hybridization, wherein, the variation of described generation signal is to produce by the insertion etc. by hybridization.In order to detect the hybridization product effectively, preferably the nucleic acid in nucleic acid molecule or the test sample is incorporated into solid phase.Such detection can be implemented as the base sequencing technologies based on such as southern blotting, microarray method, microballon method (flow cytometry) or the chromatographic nucleic acid hybridization of nucleic acid (DNA).Nucleic acid molecule can adopt the form of probe.

Under the situation of above-mentioned (2), for example, nucleic acid molecule is played a role as primer, and with the DnaJ gene as template, induce the nucleic acid molecule that makes with DnaJ gene specific hybridization to extend, whether obtain specified amplified production by detections such as electrophoresis.Such gene amplification and detection can be by implementing such as PCR, RT-PCR method, transcriptive intermediate TRAP (TMA method), branched DNA method (bDNA method), PCR in real time method or the gene amplification method of ligase chain reaction method (LCR method) or the detection method of amplified production.The method of use PCR will be explained below in the book and is described in detail.

The method of above-mentioned (3) can be implemented by combination above-mentioned (2) and above-mentioned (1).For example, can specific primer be arranged as nucleic acid molecule by using genus or bacterial classification to certain limit, obtain gene amplification product, and also can use specified strain is had specific probe as nucleic acid molecule, the gene amplification product that is produced is detected amixia.

(authentication method of bacterium)

The authentication method of bacterium of the present invention can may further comprise the steps: obtain at least a portion base sequence such as the DnaJ gene in the test sample of the clinical sample that may contain bacterium to be identified or its nucleic acid.By obtaining at least a portion base sequence of DnaJ gene, may identify the bacterial classification of bacterium to be identified.In addition, can make its phylogenetic tree.In the method for detecting bacterium of the present invention, preferably comprise streptococcus (Streptococcus) bacterium as the bacteria culture that detects target, Staphylococcus (Staphylococcus) bacterium, enterococcus spp (Enterococcus) bacterium, Peptostreptococcus (PeptoStreptococcus) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, enterobacter (Enterobacter) bacterium, legionella (Legionella) bacterium, Neisseria (Neisseria) bacterium, bacillus (Bacillus) bacterium, Helicobacterium (Helicobacter) bacterium, Corynebacterium (Corynebacterium) bacterium, Rhodopseudomonas (Pseudomonas) bacterium, Bai Huoerde Bacillaceae (Burkholderia) bacterium, Haemophilus spp (Haemophilus) bacterium, Bacteroides (Bacteroides) bacterium, enterococcus spp (Enterococcus) bacterium, Nuo Kaer Pseudomonas (Nocardia) bacterium, general Bordetella (Prevotella) bacterium, Neisseria (Neisseria) bacterium, Moraxella (Moraxella) bacterium, Flavobacterium (Flavobacterium) bacterium, fusobacterium (Clostridium) bacterium, treponema (Treponema) bacterium, Sphingobacterium (Sphingobacterium) bacterium, leptospira (Leptospira) bacterium, campylobacter (Campylobacter) bacterium, Listera belongs to (Listeria) bacterium, salmonella (Salmonella) bacterium and Yersinia (Yersinia) bacterium.Because the DnaJ gene is multifarious gene in these bacteriums, so it is for identifying that bacteria culture is useful.When obtaining the base sequence of DnaJ gene, also can obtain above-mentioned nucleic acid molecule used as primer and the base sequence of the pcr amplification product that obtains or above-mentioned nucleic acid molecule used as probe and the base sequence of the hybridization product that obtains.

Can be by obtaining base sequence, to at least a portion of the gene that obtains by PCR method etc., utilize the sequencer that adopts known method to carry out sequencing measure base sequence method or with the method (utilizing microarray method etc.) of probe hybridization with specified base sequence.

When identifying bacterial classification, preferably the base sequence that obtains and the evaluation that obtains the in advance base sequence to the DnaJ gene of bacteria-like is compared based at least a portion base sequence of the DnaJ gene that from test sample, obtains.That is, when identifying bacteria culture, preferably at first prepare the database of all or part of base sequence of the DnaJ gene of each bacteria culture.Prepare such database in advance, can promptly identify bacteria culture.In addition, preferred preparation in advance comprises the database of information of the base sequence of the DnaJ gene that relates to the various bacterial strains in the bacteria culture.Utilize such database, can easily extract the specific base sequence of the general bacterial classification of bacterial strain.By using as above operation to extract the base sequence that obtains, can be easily and identify bacteria culture exactly.In addition, also being suitable for manufacturing system simultaneously sets.

Especially, when utilizing DnaJ gene identification bacteria culture, the step of base sequence that at first obtains the 16S rDNA gene of test sample is useful.This is because even the similarity height of the base sequence of 16SrDNA gene and nearly edge bacterial classification, the similarity of DnaJ gene is also low.For example, (such bacterial classification can be a kind to the highest candidate's bacterial classification of similarity by at first measuring 16S rDNA gene, also can be) for more than 2 kinds, the base sequence of the DnaJ gene of the base sequence of the DnaJ gene of candidate's bacterial classification and strain isolated etc. in the determination test sample then can be easily and promptly identify the bacterial classification of strain isolated.In addition, in the time of can identifying by 16S rDNA gene, identify by the DnaJ gene with regard to not needing.When adopting such method, though be not particularly limited with the similarity of the 16S rDNA gene of existing bacterial classification, when the similarity of the base sequence at least 98% of the 16S of strain isolated and existing bacteria culture rDNA gene, preferably the base sequence to the DnaJ gene of this existing bacterial isolates and strain isolated compares.

When obtaining the base sequence of 16S rDNA gene, same with the base sequence that obtains the DnaJ gene, can suitably use known method.In addition, preferably compare, obtain the similarity level between these base sequences by base sequence with the 16S rDNA gene of at least a portion base sequence of the 16S rDNA gene in the test sample and existing bacterial isolates.The database of base sequence that need prepare the 16S rDNA gene of various bacteria cultures or bacterial strain in advance.

During the base sequence of the DnaJ gene in the analytical test sample etc.,, can use the aforesaid for example test sample of clinical sample that comprises though the sampling source of bacterium to be identified is not particularly limited.In addition, same with the detection method of bacterium described above, can be with the amplified production of the designated area of the nucleic acid extractive of bacterium or bacterial gene as test sample.

In addition, from the above, according to the present invention, a kind of Bacteria Identification program that is used to obtain at least a portion base sequence of DnaJ gene can also be provided, wherein, with the bacterium similar to the bacterium in the above-mentioned authentication method as bacterium to be identified, on the computer more than 1 or 2, carry out following steps, the step of the bacterial classification of the bacterium in the described test sample is identified in the i.e. base sequence contrast of the DnaJ gene of at least a portion base sequence by the DnaJ gene that will obtain from the test sample of the nucleic acid that may contain bacterium to be identified and bacterium to be identified.According to this program, can be by the sequence sequence consistent of the retrieval DnaJ gene that can from test sample, obtain from the sequence library of DnaJ gene with base sequence, thus the kind of the bacterium of qualification test sample.In this program, before above-mentioned steps, can also carry out following steps, the base sequence of at least a portion base sequence of the 16S rDNA gene that soon obtains from above-mentioned test sample and the 16S rDNA gene of bacterium to be identified compares, determine to have the candidate's bacterial classification with the base sequence of the highest 16S rDNA gene of the base sequence similarity of gained, and the base sequence of the DnaJ gene of these candidate's bacterial classifications and base sequence from the DnaJ gene of test sample are compared.

Here, the form of program is not special considers.Program can be the form of downloading by the Internet, also can be for being stored in the form in the suitable storage media.Perhaps, described program can also be stored among the ROM etc. of computer or other appropriate device.And, according to the present invention, can also provide the device of storing such program in the mode that can carry out.The controller of this device can have the CPU that can carry out such program, store the device such as the storing device of ROM or hard disk and suitable list entries of this program.In addition, can also be in the storing devices such as hard disk in the device sequence library of equipment DnaJ gene, and, if be the environment that can connect the Internet, then can also carry out necessary processing by the server that visit stores such sequence library.And the step of contrast sequence is promptly measured the similarity level between sequence or the step of comparing can be used all or part of of DNASIS pro version (Hitachi's software), Beacon designer version 5.1 (Molecular Beacon company) and Clustal W softwares such as (free softwares).

(nucleic acid molecule and comprise detection and the identification kit of the bacterium of nucleic acid molecule)

Nucleic acid molecule of the present invention can comprise above-mentioned various types of nucleic acid molecule.In addition, can also be as being used to detect or identifying that the detection and the identification kit that comprise the bacterium of above-mentioned various nucleic acid molecule more than a kind or 2 kinds prepare such nucleic acid molecule.The selected contained nucleic acid molecule of such detection and identification kit that is used for can be the nucleic acid molecule that works as probe and/or primer.Detect and identification kit in the combination of nucleic acid molecule be not particularly limited, though test kit be preferably combination have can detect or identify preferred more than 3 kinds, more preferably more than 4 kinds, the test kit of the nucleic acid molecule of further preferred bacteria culture more than 5 kinds.The example of the combination of preferred target bacterial classification is had a liking for chlamydiaceae (Chlamydophila) bacterium for comprising 2 kinds, 1 kind of chlamydiaceae (Chlamydia) bacterium, 1 kind of Coxiella (Coxiella) bacterium, 1 kind of Haemophilus spp (Haemophilus) bacterium, 4 kinds of legionellas (Legionella) bacterium, 2 kinds of streptococcuses (Streptococcus) bacterium, 3 kinds of Mycobacteriums (Mycobacterium) bacterium, 1 kind of Rhodopseudomonas (Pseudomonas) bacterium, 1 mycoplasma species belongs to (Mycoplasma), 1 kind of Staphylococcus (Staphylococcus) bacterium, 1 kind of special Pseudomonas (Bordetella) bacterium of Boulder, 1 kind of Corynebacterium (Corynebacterium) bacterium, 1 kind of special Pseudomonas (Bordetella) bacterium of Boulder, 1 kind of klebsiella (Klebsiella) bacterium, the combination of a kind of Serratia (Serratia) bacterium and a kind of Escherichia (Escherichia) bacterium.The test kit of such nucleic acid molecule is preferred for detecting and identifying the pathogenic bacteria of pneumonia and pharyngitis.The example of another kind of preferably combination is for comprising the combination that a kind of chlamydiaceae (Chlamydia) bacterium, 2 kinds of Neisserias (Neisseria) bacterium, 2 mycoplasma species belong to (Mycoplasma) bacterium, 2 kinds of urea mycoplasmas (Ureaplasma) bacterium, a kind of Candida (Candida) bacterium, a kind of treponema (Treponema) bacterium, a kind of Trichomonas (Trichomonasa) bacterium and 2 kinds of Haemophilus spps (Haemophilus) bacterium.The test kit of such nucleic acid molecule is preferred for detecting and identifying the pathogenic bacteria of venereal disease and urinary tract infection.Other examples of preferably combination are for comprising 2 kinds of Yersinias (Yersinia) bacterium, 4 kinds of Escherichias (Escherichia)-Shigella (Escherichia-Shigella) bacterium, 1 kind of campylobacter (Campylobacter) bacterium, 1 kind of Salmonella (Salmonella) bacterium, 4 kinds of Vibrios (Vibrio) bacterium, 3 kinds of Aeromonass (Aeromonas) bacterium, 1 kind of Plesiomonas (Plesiomonas) bacterium, 1 kind of giardia (Giardia) bacterium, the composition of a kind of entamoeba (Entamoeba) bacterium and 4 kinds of fusobacteriums (Clostridium) bacterium.The test kit of such nucleic acid molecule is preferred for detecting and identifying the pathogenic bacteria of food poisoning and diarrhoea.

More than preferred combination also can with particularly with 16S rDNA as target, the product that the nucleic acid in the test sample increases is identified that the method for bacteria culture is used in combination by PCR etc.For example, can utilize the primer mixture (composition) of the primer sets that comprises the 16S rDNA gene that to increase, also can at first attempt identifying bacterial classification, under situation about can not identify like this, reattempt by DnaJ and identify by the mode of 16S rDNA.Thus, can detect simultaneously or identify a plurality of bacteria cultures, or detect or identify bacteria culture effectively.

Such detection and identification kit can only be prepared as and for example comprise a kind or 2 kind or more the test kit of nucleic acid molecule of nucleic acid molecule as the form of the gene amplification of primer, can be prepared as also wherein that nucleic acid molecule can be fixed in such as the test kit on the solid phase of microballon or matrix as probe more than a kind or 2 kinds.

(immobilization body)

Immobilization body of the present invention can obtain by the solid phase that is fixed in such as microballon or matrix more than a kind or 2 kinds with above-mentioned nucleic acid molecule.The nucleic acid molecule that is fixed on the solid phase is identified by the positional information of its sequence in matrix etc. or by its color in the microballon.The nucleic acid molecule that has been fixed on the solid phase also can be in conjunction with the compound that transmits after the hybridization on it.In addition, nucleic acid molecule can be fixed in and can carry out primer prolongation reaction on the solid phase surface.The form of solid phase is not particularly limited, and its material also is not particularly limited, and for example, can use glass, plastics or pottery.

In addition, immobilization body of the present invention can be for being fixed in the nucleic acid chromatograms device that obtains on the chromatogram base material such as strainer as the nucleic acid molecule of probe with mistake.

The invention still further relates to the amplification method and the application thereof that are used to increase or detect at least a kind of target nucleic acid.These inventions are except can be used for amplification and detect nonspecific target nucleic acid, also can be used for above-mentioned DnaJ gene or its part utilizing the Bacteria Detection and the evaluation of the base sequence of DnaJ gene as target nucleic acid.That is,, can improve the present invention further to (preferred more than the 2 kinds) detection of bacteria culture and availability of evaluation more than a kind or 2 kinds by using bacteria culture Auele Specific Primer based on the base sequence of DnaJ gene.

The amplification method of target nucleic acid of the present invention is the method for at least a kind of target nucleic acid of amplification, this method can may further comprise the steps, promptly, use at least a kind of the 1st primer sets that has flag sequence respectively and described target nucleic acid is had specific base sequence, with at least a kind of the 2nd primer sets of use, implement the step of polymerase chain reaction with flag sequence substantially the same with the flag sequence of described the 1st primer sets.

Utilize target nucleic acid amplification method of the present invention,, comprise the amplified production of the 1st primer sets acquisition of target nucleic acid sequence by the amplification of the 2nd primer sets by the 1st primer sets amplifying target nucleic acid.That is,, utilize the 2nd primer sets to increase, can easily obtain to comprise the amplified production of the target nucleic acid that detects needed amount even utilize the efficient of the 1st primer sets amplifying target nucleic acid low.Therefore, even multiple the 1st primer sets is a lower concentration, also can be by remedying the poor efficiency of utilizing the 1st primer sets to increase, and utilize the 1st primer sets above target nucleic acid that can increase.In addition, owing to mainly utilize the 2nd primer sets to come amplifying target nucleic acid, therefore, can make the easy homogeneous of amplification efficiency.

Can make the step of above-mentioned polymerase chain reaction step for the amplified production implementing to utilize above-mentioned the 1st primer sets simultaneously and increase described target nucleic acid and utilize described the 2nd primer sets amplification to utilize described the 1st primer sets to increase to obtain.Such polymerase chain reaction step can easily detect target nucleic acid.

Among the present invention, the target nucleic acid that the 1st primer sets can be designed to have on the DnaJ gene of microorganism has specific base sequence.By use DnaJ gene, though the microorganism that existence does not belong to together and same genus is not of the same race, also can be easily in bacterial classification level detection microorganism.The inventor is clear and definite, and it is low that the DnaJ gene has between bacterial classification similarity, but at the conservative base sequence of same bacterial classification.Therefore, the DnaJ gene pairs is favourable because of the detection of the different pathogenic micro-organism of bacterial classification difference toxicity or grade of malignancy level.In addition, by using the DnaJ gene, amplified production easily can be designed the length below 250 bases of the competition that can effectively avoid amplified production.

The embodiments of the present invention of the figure that following reference is suitable are elaborated to the amplification method of target nucleic acid of the present invention and the test kit that is used for this amplification.Figure 14 has shown the 1st primer sets used in the amplification method of the present invention and the relation of the 2nd primer sets and target nucleic acid.

(polymerase chain reaction)

Among the present invention, polymerase chain reaction (PCR) is in vitro by the reaction of enzyme reaction with specific dna sequence dna amplification.Existing PCR method comprises RT-PCR method, PCR in real time method, and various improved method and with other the combination of analytical technology, can comprise these all methods among the PCR that the present invention uses.

(target nucleic acid)

Among the present invention, term " target nucleic acid " is meant the whole of the base sequence that increases by polymerase chain reaction or the nucleic acid that it is a part of.Target nucleic acid is so long as can just be not particularly limited by the target nucleic acid that polymerase chain reaction increases, and example comprises that except as genome, vectorial DNA of organism genetic information or RNA also have mRNA, cDNA and cRNA.In addition, target nucleic acid can be strand or double-stranded.

Target nucleic acid can extract from organism or one portion of tissue and obtain and can be various ways, such as the amplified production by obtaining in the enterprising performing PCR reaction of the mRNA that expression analysis is obtained, cDNA and genome, there is no particular limitation for described form.As the organism in its source or organize also and be not particularly limited, except that the animal that comprises the mankind and non-human mammal, also can be used as the source such as the microorganism of bacterium, fungi and virus.Consider that from the prevention of infecting disease and the viewpoint of treatment and health the source organism of target nucleic acid is preferably microorganism, more preferably because health or treatment, the detected or microorganism of identifying, more preferably pathogenic micro-organism.

The example of the microorganism that target nucleic acid is originated comprises Staphylococcus (Staphylococcus) bacterium, streptococcus (Streptococcus) bacterium, Serratia (Serratia) bacterium, klebsiella (Klebsiella) bacterium, Escherichia (Escherichia) bacterium, Mycobacterium (Mycobacterium) bacterium, legionella (Legionella) bacterium, Vibrio (Vibrio) bacterium, bacillus (Bacillus) bacterium, Neisseria (Neisseria) bacterium, campylobacter (Campylobacter) bacterium, chlamydiaceae (Chlamydia) bacterium, have a liking for chlamydiaceae (Chlamydophila) bacterium, Mycoplasma (Mycoplasma) bacterium, Listera belongs to (Listeria) bacterium, salmonella (Salmonella) bacterium and Yersinia (Yersinia) bacterium.The microorganism of the bacteria culture more than a kind or 2 kinds is selected from the bacterium that belongs to these genus.The bacterial classification that belongs to these genus has the preferred target nucleic acid on the DnaJ gene.This is because in belonging to the microorganism of these genus, the similarity of the base sequence of the DnaJ gene between bacterial classification is lower than the similarity of the base sequence of 16S rDNA gene, therefore finds bacterial classification specificity base sequence conservative between the bacterial strain of same bacterial classification easily.

Wherein, be preferably the bacteria culture of Staphylococcus (Staphylococcus), streptococcus (Streptococcus), Mycobacterium (Mycobacterium), legionella (Legionella) and Vibrio (Vibrio).This is because the similarity of the bacterium that belongs to these genus DnaJ gene between belonging to and between the bacterial classification in the same genus is low, therefore is suitable for the mode bacterial detection bacterial classification by the DnaJ gene.For example, for the DnaJ gene, in Staphylococcus (Staphylococcus) bacterium, the similarity average out to 97.4% of 16S rDNA gene between bacterial classification, the similarity average out to 77.6% of DnaJ gene between bacterial classification, in the bacterium of streptococcus (Streptococcus), the similarity average out to 95.8% of 16SrDNA gene between bacterial classification, the similarity average out to 76.4% of DnaJ gene between bacterial classification, in Mycobacterium (Mycobacterium) bacterium, the similarity average out to 93% of 16S rDNA gene between bacterial classification, the similarity average out to 83.7% of DnaJ gene between bacterial classification, in the bacterium of legionella (Legionella), the similarity average out to 96.1% of 16S rDNA gene between bacterial classification, the similarity average out to 82.5% of DnaJ gene between bacterial classification, for the bacterium of Vibrio (Vibrio), the similarity average out to 97.4% of 16S rDNA gene between bacterial classification, the similarity average out to 82% of DnaJ gene between bacterial classification.

Such target nucleic acid with blood for example, serum, from comprise cell or tissue that the people collects interior animal, the mode of test samples such as movement, phlegm, well water, food such as ight soil and urine provides.Test sample can be the nucleic acid extraction sample that obtains by the material extraction nucleic acid from such collection, perhaps can be the sample of the amplified production that comprises DnaJ gene or its part (comprising zone to be detected).

(the 1st primer sets)

Use in the amplification method of the present invention and have flag sequence and selectively annealed the 1st primer sets that arrives the base sequence of target nucleic acid.The 1st primer sets is made of forward primer and reverse primer.As shown in Figure 1, forward primer and reverse primer all have flag sequence in its 5 ' side, have the sequence that is annealed to target nucleic acid in 3 ' side.

The 1st primer sets (primer to) is selected according to the kind of target nucleic acid.Usually, select single the 1st primer sets with respect to single target nucleic acid.Yet, also can select single the 1st primer sets sometimes to two above target nucleic acids.

Selectively annealed to the 1st primer sets (to) base sequence of separately the target nucleic acid that primer had (below be also referred to as anneal sequence) is preferred only selects and target nucleic acid hybridization, sequence that the specificity of can annealing is high.Here, will be for same gene design during as the primer of target nucleic acid, between the different primer sets, anneal sequence can partly or entirely be mated.Anneal sequence can be according to the specificity of target nucleic acid and avoid the formation of primer dimer, the temperature of reaction in the PCR step to determine.Temperature of reaction Tm between preferred this base sequence and the target nucleic acid is more than 50 ℃ below 70 ℃, more preferably more than 55 ℃ below 65 ℃.

With the detection of microorganism or be accredited as purpose and when implementing amplification method, such anneal sequence can select that microorganism to be detected or that identify is had specific sequence, though, preferably use base sequence on the DnaJ gene as target nucleic acid as mentioned above.For a plurality of microorganisms, with the base sequence of same gene during as target nucleic acid, the base sequence of DnaJ gene that can be by a plurality of microorganisms of comparison is selected microorganism specificity base sequence.Such anneal sequence is that bacterial classification is specific, and can detect the bacterial strain that belongs to this bacterial classification reliably.For the such comparison and the extraction of characteristic sequence, can utilize software such as DNASIS pro version (Hitachi's software), Beacon designer 5.1 versions (Molecular Beacon company) and Clustal W (free software).

Flag sequence can be selected from not the sequence with target nucleic acid to be amplified hybridization.The length of flag sequence is preferably below above 40 bases of 12 bases, more preferably below above 30 bases of 18 bases.

Constitute the forward primer of the 1st primer sets and reverse primer and can have joint sequence between flag sequence and anneal sequence respectively, though preferably only constitute by 2 kinds of sequences.In addition, all preferred integral body of forward primer and reverse primer sequence is below above 80 bases of 30 bases, more preferably more than 35 bases.

In addition, utilize the 1st such primer sets and the amplified production that obtains that increases is preferably the base below 280 more than 80.This is because in the time of in this scope, the possibility of the general conserved sequence that comprises in the gene of pathogenic micro-organism etc. reduces, the detection sensitivity raising.More preferably amplified production is below 250 bases.This be because, be 250 bases when following, the possibility of the general conserved sequence of the different bacterium bacterial classification that amplification region comprises reduces, amplification efficiency and sensitivity raising.Further preferred amplified production is below 200 bases.This be because, be 200 bases when following, design bacterial classification Auele Specific Primer and probe easily.Particularly with the DnaJ gene of microorganism during as target nucleic acid, the structure of DnaJ gene, when the length of amplified production surpassed 200 bases, it is difficult that the design of bacterial classification Auele Specific Primer and probe becomes, and is 200 bases when following, and the design transfiguration of bacterial classification Auele Specific Primer is easy.In addition, during for the DnaJ gene, the structure of gene, even amplified production is this length, the design of specific probe is also easy than 16S rDNA.That is, by on the DnaJ gene, disposing target nucleic acid, can in a plurality of microorganisms, design can obtain below preferred 250 bases, more preferably the 1st primer sets of the amplified production below 200 bases.In the present invention, because the base length of certain limit that can be such obtains the product that utilizes the 1st primer sets to increase, therefore, can be used to utilize the 2nd primer sets further increase this amplified production the PCR circulation and utilize the PCR reaction cycle of the 1st primer sets to be set at same circulation.

Use at least a the 1st primer sets, amplification method of the present invention can use multiple the 1st primer sets when the amplification step of utilizing multiplex PCR to implement etc.Preferably use the 1st primer sets more than 6 kinds, more preferably use the 1st primer sets more than 10 kinds.Even more preferably use the 1st primer sets more than 15 kinds.Yet, preferably use the 1st primer sets below 50 kinds.When the target nucleic acid of amplification microorganism, preferred especially with the sequence on the DnaJ gene as target nucleic acid.By using this gene, can design the primer that is difficult to compete between the special and amplified production of bacterial classification, thereby can easily detect the target nucleic acid of multiple microorganism by multiplex PCR.

(the 2nd primer sets)

In this amplification method, can use at least a kind of the 2nd primer sets with flag sequence substantially the same with the flag sequence that uses in the 1st primer sets.The amplified production annealing of the 2nd primer sets by obtaining by flag sequence and the 1st primer sets and target nucleic acid annealing can further be increased amplified production.

The 2nd forward primer and the 2nd reverse primer that constitute the 2nd primer sets have respectively and the flag sequence of the 1st forward primer and the substantially the same flag sequence of flag sequence of the 1st reverse primer.The 2nd forward primer and the 2nd reverse primer preferably comprise respectively the substantially the same flag sequence of flag sequence with the 1st forward primer and the 1st reverse primer.

Here, the flag sequence substantially the same with flag sequence for example is the flag sequence identical with the flag sequence of the 1st forward primer, the base sequence of modifying in can keeping the specific scope of hybridization of the 1st flag sequence and complementary base sequence.Illustrative examples comprises and this base sequence complementary base sequence, or in this base sequence, modifies and the base sequence that obtains in the mode more than a kind or 2 kinds in the base more than 1 or 2 of being selected from displacement, inserting, lacking and adding.Preferred use be added on the 1st primer sets respectively on the identical flag sequence of flag sequence.

Not corresponding the 1st specific primer sets of the 2nd primer sets is used, but uses corresponding to the flag sequence that can use in the 1st primer sets.Therefore, the 1st primer sets of in amplified reaction, using more than 2 kinds use a kind of flag sequence to the time, the 2nd primer sets is a kind.Perhaps, when the 1st primer sets more than 2 kinds is used flag sequence more than 2 kinds, use the 2nd primer sets more than 2 kinds.

Preferred so that each primer concentration of the 1st primer sets each primer concentration low, that make the 2nd primer sets mode higher than each primer concentration of the 1st primer sets of the 1st primer sets like this and the 2nd primer sets used.This be because, owing to use the 2nd primer sets, therefore, as long as can produce can be as the amplified production of the degree of the template of the 2nd primer sets for the 1st primer sets.

The concentration of each primer of the 2nd primer sets in the PCR reaction soln is preferably more than 10 times below 50 times of concentration of each primer of each the 1st primer sets in the PCR reaction soln.In the time of in this scope,, also can obtain similar detection sensitivity when increasing with independent primer sets even use multiple the 1st primer sets.Here, the ratio of the primer concentration of the primer concentration of the 1st primer sets and the 2nd primer sets preferably decides according to the species number of employed the 1st primer sets.For example, when the kind of employed the 1st primer sets is 10 kinds, preferably the concentration of each primer of the 2nd primer sets is made as more than 10 times of primer concentration of each the 1st primer sets.Equally, when the kind of employed the 1st primer sets is 20 kinds, preferably the concentration of each primer of the 2nd primer sets is made as more than 20 times of primer concentration of each the 1st primer sets.

The concentration of each primer of the 1st primer sets is preferably below the above 0.2 μ M of 0.005 μ M.In the time of in this scope, can utilize the 2nd primer sets to produce amplified production with the level that can increase.More preferably primer concentration is below the 0.1 μ M.On the other hand, the primer concentration of the 2nd primer sets is preferably more than the 0.1 μ M, more preferably more than the 0.25 μ M.

The 1st primer sets of using among the present invention and the 2nd primer sets preferably possess any in the following feature:

(a): utilize described the 1st primer sets to increase and the product that obtains is to have 250 nucleic acid with next base

(b): the concentration the when primer of described the 1st primer sets uses is the above 0.2 μ M of 0.005 μ M following (preferred 0.1 μ M is following); Perhaps

(c): the concentration the when primer of described the 2nd primer sets uses is more than 10 times below 50 times of the concentration of the primer of described the 1st primer sets when using.

By possessing these features,, also can keep the target sequence of the specificity and the aequum that increases even use multiple the 1st primer sets.In addition, can also implement to use the amplification step of multiple the 1st primer sets as described later by simple PCR step.

Here, constitute the nucleic acid molecule of these the 1st primer sets and the 2nd primer sets so long as can carry out just being not particularly limited based on the nucleic acid molecule of the hybridization of base sequence.Though typical nucleic acid molecule is DNA, can adopt any form in DNA, RNA, DNA-RNA mosaic, the artificial nucleic acid that contains modified base or the peptide nucleic acid(PNA) (PNA).In addition, such nucleic acid molecule can also be used multiple fluorescence dye, and the quencher of fluorescence dye etc. is modified.

(PCR step)

Use reaction conditions in the PCR step of the 1st primer sets and the 2nd primer sets etc. to be not particularly limited, as long as can utilize the amplification of the 2nd primer sets to utilize the amplified production of the target nucleic acid that the 1st primer sets increases.Utilize the amplification step of the target nucleic acid of the 1st primer sets can implement with the circulation of different " sex change-annealing-extension " respectively with the step that the amplified production that utilizes the 2nd primer sets to the 1st primer sets increases.For example, utilize the amplification step of the 1st primer sets can be,, implement in 1 second～30 seconds order below 75 ℃ more than 70 ℃ at more than 55 ℃ below 65 ℃ 10 seconds～60 seconds at more than 90 ℃ below 97 ℃ 10 seconds～60 seconds.In addition, for example, cycle number be preferably 1 circulation above several below circulation.This be because, the circulation above 10, the increase degree of the amount of final amplified production stops to become greatly.Be preferably 1 or 2 circulation, more preferably 1 circulation.In addition, for example, utilize the amplification step of the 2nd primer sets implementing 0.5 second～5 seconds below 97 ℃ more than 90 ℃, implementing 0.5 second～5 seconds below 65 ℃ more than 55 ℃, implementing 1 second～20 seconds below 75 ℃ more than 70 ℃.In addition, cycle number is preferably below above 70 circulations of 20 circulations.More preferably more than 30 circulations, more preferably below above 60 circulations of 40 circulations.

In addition, utilize the amplification step of the 1st primer sets and utilize PCR that the amplification step of the 2nd primer sets can single " sex change-annealing-extension " to circulate and implement.The length setting of the amplified production that will obtain utilizing the 1st primer sets to increase is below preferred 250 bases, when more preferably 200 bases are following, and the condition enactment that especially preferably will utilize the PCR reaction cycle of the 2nd primer sets is and the identical condition of PCR circulation of utilizing the 1st primer sets.Under these circumstances, can the 1st primer sets and the 2nd primer sets be played a role the PCR circulation by identical conditions effectively respectively, its result can obtain utilizing the amplified production of the 2nd primer sets effectively.Therefore, there is no need to implement in addition to be used for the PCR circulation of the 1st primer sets.

As previously discussed, by target nucleic acid being placed on the DnaJ gene, the length of the amplified production that will obtain when utilizing a plurality of the 1st primer sets that a plurality of target nucleic acids are increased is below 250 bases, when preferred 200 bases are following, implements single PCR circulation time, especially preferably implements multiplex PCR.Design on such base length by amplified production the 1st primer sets, not only can when utilizing the 1st primer sets to increase, suppress competition, and owing to can give impartial amplification condition to all target nucleic acids, therefore arbitrary nucleic acid all can identical detection sensitivity detect.In addition, by in single PCR circulation, the 1st primer sets and the 2nd primer sets all being played a role, also be suitable for quantitative analysis such as PCR in real time.

Use single PCR circulation, when utilizing the 1st primer sets and the 2nd primer sets to implement amplification step, can be at more than 90 ℃ below 97 ℃ 0.5 second～5 seconds, at more than 55 ℃ below 65 ℃ 0.5 second～5 seconds, implement in 1 second～20 seconds order below 75 ℃ more than 70 ℃.In addition, cycle number is preferably below above 70 circulations of 20 circulations.Below above 60 circulations of 30 circulations more preferably.

In addition, in such PCR step, the primer concentration of the 1st primer sets is preferably below the above 0.2 μ M of 0.005 μ M, and the primer concentration of described the 2nd primer sets is preferably more than 10 times below 50 times of primer concentration of described the 1st primer sets.In the time of in such concentration range, amplifying target nucleic acid effectively.In addition, utilize the 1st primer sets to increase and during the amplified production that the obtains nucleic acid that to be 250 bases following, the possibility that comprises the zone with similar sequences reduces, and can increase sensitivity under the situation of the competition that does not cause amplification.

When the present invention is applicable to the detection method of previously described bacteria culture, preferably described (a) step is set at following steps, promptly, use at least a kind of the 1st primer sets with base sequence of a flag sequence and a selectively annealed part to described DnaJ gene, at least a kind of the 2nd primer sets with flag sequence substantially the same with the described flag sequence of the 1st primer sets, utilize described the 1st primer sets increase described DnaJ gene at least a portion and utilize the 2nd primer sets amplification to utilize described the 1st primer sets to increase and the step of the amplified production that obtains is implemented polymerase chain reaction.Thus, even when the possibility that has a plurality of bacteria cultures is arranged, also can a few experiments detect a plurality of bacterial classifications widely and whether exist.Under this situation,, experiment further can be simplified by the PCR step being set at the step of implementing single PCR reaction cycle.

(utilizing the method for multiplex PCR diagnosis pathogenic micro-organism)

Method based on multiplex PCR diagnosis pathogenic micro-organism of the present invention is thin to be selected from Staphylococcus (Staphylococcus), streptococcus (Streptococcus) is thin, Serratia (Serratia) is thin, klebsiella (Klebsiella) is thin, Escherichia (Escherichia) is thin, Mycobacterium (Mycobacterium) is thin, legionella (Legionella) is thin, Vibrio (Vibrio) is thin, bacillus (Bacillus) is thin, Neisseria (Neisseria) is thin, campylobacter (Campylobacter) is thin, chlamydiaceae (Chlamydia) is thin, it is thin to have a liking for chlamydiaceae (Chlamydophila), Mycoplasma (Mycoplasma) is thin, it is thin that Listera belongs to (Listeria), the pathogenic micro-organism more than 2 kinds that Salmonellas (Salmonella) carefully reaches Yersinia (Yersinia) detail is an object, this method possesses uses at least a the 1st primer sets and at least a the 2nd primer sets to containing the step of implementing polymerase chain reaction from the test sample of the nucleic acid of described pathogenic micro-organism, contain the step of the amplified production of described target nucleic acid with detection, described the 1st primer sets has the base sequence of flag sequence and selectively annealed target nucleic acid to the DnaJ gene that described pathogenic micro-organism had, and described the 2nd primer sets has the flag sequence substantially the same with the flag sequence of described the 1st primer sets.The PCR step can be implemented in operation similar to the above.Especially preferably with the step of PCR step for the single PCR reaction cycle of enforcement.Thus, can promptly diagnose pathogenic micro-organism.Detect in the step, the method that detects amplified production is not particularly limited.For example, can separate based on molecular weight such as electrophoretic technology by use and detect amplified production, also can make the 2nd primer linkage flag compound, utilize Southern blotting, microarray method, microballon method (flow cytometry), nucleic acid (DNA) chromatography to detect target nucleic acid then based on hybridization.

According to diagnostic method of the present invention, by having described PCR step, even only there is a spot of target nucleic acid, also can be special and detection sensitivity detect target nucleic acid well.Even use multiple primer sets, when utilizing multiplex PCR to detect target nucleic acid, can specificity good, sensitivity well and easily detects target nucleic acid.In addition, because target nucleic acid is on the DnaJ gene, therefore, be elected to be anneal sequence in the sequence that will on the DnaJ of different microorganisms gene, guard as much as possible, when designing the 1st primer sets, the target nucleic acid on the DnaJ gene that the 1st primer sets increases different equably, increase the equably amplified production of gained of the 2nd primer sets, therefore amplifying target nucleic acid can quantifying target nucleic acid be the amount of microorganism quantitatively.This kind quantitative analysis also goes for other target nucleic acid.For example, also be applicable to target nucleic acid such as mDNA or cDNA as the product of expression analysis.

(test kit that comprises the primer sets that is used for the target nucleic acid by at least a kind of pathogenic micro-organism of multiplex PCR amplification)

Test kit of the present invention can comprise the 1st primer sets and the 2nd primer sets, described the 1st primer sets has the base sequence of flag sequence and selectively annealed target nucleic acid in described pathogenic micro-organism, and described the 2nd primer sets has the flag sequence substantially the same with the flag sequence of described the 1st primer sets.The 1st primer sets in this detection kit and the 2nd primer sets all can be suitable for the 1st primer sets in the amplification method of the present invention that has illustrated and the embodiment of the 2nd primer sets.

And test kit of the present invention preferably possesses any in the following feature, described being characterized as:

(a): utilize described the 1st primer sets to increase and the product that obtains is the following nucleic acid of 250 bases

(b): the concentration the when primer of described the 1st primer sets uses is the above 0.2 μ M of 0.005 μ M following (preferred 0.1 μ M is following); Perhaps

(c): the concentration the when primer of described the 2nd primer sets uses is more than 10 times below 50 times of the concentration of the primer of described the 1st primer sets when using.

By possessing these features, not only can easily increase or detect a spot of target nucleic acid, even and use multiple primer right simultaneously, also can be reliably and detect target nucleic acid in high sensitivity.

Test kit of the present invention preferably has target nucleic acid on the DnaJ gene.By on the DnaJ gene, having target nucleic acid,, also can design not amplified production with other amplified production competition even the product that utilizes the 1st primer sets to increase is the following nucleic acid of 200 bases.This detection kit is preferred for the target nucleic acid on the DnaJ gene of microorganism.As the explanation of front, when these microorganisms have target nucleic acid on the DnaJ gene of any microorganism shown in the following table, preferably use forward primer shown in the same table and reverse primer.

Table 1

Mycobacterium tuberculosis (Mycobacterium tuberculosis) DnaJ	The pneumonia primers F	5-TAGCAGGATCCCTCTAAG-TTGTT CGGTGGCTTGTTC
			Mycobacterium tuberculosis (Mycobacterium tuberculosis) DnaJ	Pneumonia primer R	5-AATTCTAATACGACTCACTATAG GGAG-CCTCCACGAAATCCAACTC
Intestinal bacteria (Escherichia coli) DnaJ	The pneumonia primers F	5-TAGCAGGATCCCTCTAAG-TGTT GAGCGCAGCAAAAC
			Intestinal bacteria (Escherichia coli) DnaJ	Pneumonia primer R	5-AATTCTAATACGACTCACTATAG GGAG-CAAGACGGATGCGGTCTC

Diplococcus gonorrhoeae (Neisseria gonorrhoeae) DnaJ	The STD primers F	5-TAGCAGGATCCCTCTAAGTTGGA TTTGCATTGCGAACTG
			Diplococcus gonorrhoeae (Neisseria gonorrhoeae) DnaJ	STD primer R	5-AATTCTAATACGACTCACTATAG GGAG-CCAAGGTCGGCACTTCTAA C
Vibrio cholerae (Vibrio cholerae) DnaJ	The diarrhoea primers F	5-TAGCAGGATCCCTCTAAGCAGGT TTGYTGCACGGCGAAGA
			Vibrio cholerae (Vibrio cholerae)	Diarrhoea primer R	5-AATTCTAATACGACTCACTATAG GGAG-AGAAGCGGTTCGCGGCTGC TCG
Vibrio parahaemolyticus (Vibrio parahaemolyticus) DnaJ	The diarrhoea primers F	5-TAGCAGGATCCCTCTAAGCAGGT TTGYTGCACGGCGAAGA
			Vibrio parahaemolyticus (Vibrio parahaemolyticus)	Diarrhoea primer R	5-AATTCTAATACGACTCACTATAG GGAG-GAAGCCGTTCGTGGTGTAA CT

A part of sequence of DnaJ gene is known.For with the DnaJ gene as the target nucleic acid among the present invention, need obtain in the DnaJ gene of high polymorphism at bacterial classification level conservative sequence and SNP pleomorphism site, but these sequences and site are unknown.The sequence of the DnaJ gene by measuring a large amount of wild strains such as the inventor, and identify zone that the bacterial classification specificity is conservative and the site of SNP, can at first be identified for the sequence of the specific probe of the 1st primer sets thus.In addition, can be identified for the sequence of the specific probe of amplified production.The inventor from above-mentioned content-aware for the DnaJ gene, polymorphism between the bacterial strain of the bacterial classification in Mycobacterium (Mycobacterium) is less than 1%, between the bacterial classification of intestinal bacterium section or the polymorphism between the bacterial classification of streptococcus (Streptococcus) and Staphylococcus (Staphylococcus) be 1～3%, on the other hand, the sequence difference of existence more than 5% between independently near edge bacterial classification.

Test kit of the present invention can be selected from Staphylococcus (Staphylococcus) for being used for detecting, streptococcus (Streptococcus), Serratia (Serratia), klebsiella (Klebsiella), Escherichia (Escherichia), Mycobacterium (Mycobacterium), legionella (Legionella), Vibrio (Vibrio), bacillus (Bacillus), Neisseria (Neisseria), campylobacter (Campylobacter), chlamydiaceae (Chlamydia), have a liking for chlamydiaceae (Chlamydophila), Mycoplasma (Mycoplasma), Listera belongs to (Listeria), the right test kit of primer of a kind or 2 kinds bacteria culture of Salmonellas (Salmonella) and Yersinia (Yersinia).Preferably so that a part of sequence on the DnaJ gene of these microorganisms is designed primer as the mode of target nucleic acid.Primer design can utilize amplification to belong to the bacterial classification specificity primer sequence partly of the various bacteria cultures of above-mentioned these genus.Test kit preferably combination of the present invention corresponding in these objects to be detected more than 2 kinds or more than 3 kinds the primer of each DnaJ gene of bacteria culture right.

Test kit of the present invention can be prepared as the test kit of bacteria culture more than a kind or 2 kinds that is used for detecting the various pathogenic bacterias that are selected from above-mentioned pneumonia or pharyngitis.In addition, test kit of the present invention can also be prepared as the test kit of bacteria culture more than a kind or 2 kinds that is used for detecting the various pathogenic bacterias that are selected from above-mentioned diarrhoea and food poisoning.In addition, test kit of the present invention can also be prepared as the test kit of bacteria culture more than a kind or 2 kinds that is used for detecting the various pathogenic bacterias that are selected from above-mentioned venereal disease.Test kit of the present invention preferably uses part DnaJ gene in these microorganisms to be detected as target nucleic acid.Primer design can be utilized the specific primer sequence of the various pathogenic bacterias that belong to above-mentioned these genus.Test kit preferably combination of the present invention is corresponding to the primer sets of each DnaJ gene of bacterial classification more than 2 kinds or more than 3 kinds in these microorganisms to be detected.

That test kit of the present invention can increase simultaneously is preferred more than 3 kinds, more preferably more than 4 kinds, further the mode of the target nucleic acid of preferred microorganism more than 5 kinds constitutes.For example, the combination of preferred target bacterial classification can comprise and have a liking for chlamydiaceae (Chlamydophila) in such detection kit, chlamydiaceae (Chlamydia), Coxiella belongs to, Haemophilus spp (Haemophilus), legionella (Legionella), streptococcus (Streptococcus), Mycobacterium (Mycobacterium), Rhodopseudomonas (Pseudomonas), Mycoplasma (Mycoplasma), Staphylococcus (Staphylococcus), Bordetella belongs to, Corynebacterium (Corynebacterium), Bordetella belongs to, klebsiella (Klebsiella), the bacterium of Serratia (Serratia) and Escherichia (Escherichia).Such test kit is preferred for detecting and identifying the pathogenic bacteria of pneumonia and pharyngitis.In addition, such detection kit can comprise the bacterium of chlamydiaceae (Chlamydia), Neisseria (Neisseria), Mycoplasma (Mycoplasma), urea mycoplasma (Ureaplasma), Candida (Candida), treponema (Treponema), Trichomonas (Trichomonasa) and Haemophilus spp (Haemophilus).Such test kit is preferred for detecting and identifying the pathogenic bacteria of venereal disease and urinary tract infection.Further, such detection kit can comprise the bacterium that Yersinia (Yersinia), Escherichia-Shigella (Escherichia-Shigella), campylobacter (Campylobacter), Salmonellas (Salmonella), Vibrio (Vibrio), Aeromonas (Aeromonas), Plesiomonas (Plesiomonas), giardia (Giardia), entamoeba (Entamoeba), fusobacterium (Clostridium) and Listera belong to (Listeria).Such test kit is preferred for detecting and identifying the pathogenic bacteria of food poisoning and diarrhoea.

From the above, according to the present invention, a kind of method is provided, this method is used for detection and is selected from Staphylococcus (Staphylococcus), streptococcus (Streptococcus), klebsiella (Klebsiella), Escherichia (Escherichia), Mycobacterium (Mycobacterium), legionella (Legionella), Vibrio (Vibrio), bacillus (Bacillus), Neisseria (Neisseria), campylobacter (Campylobacter), chlamydiaceae (Chlamydia), have a liking for chlamydiaceae (Chlamydophila), Mycoplasma (Mycoplasma), Listera belongs to (Listeria), the bacteria culture more than 2 kinds in the bacterium of Salmonellas (Salmonella) and Yersinia (Yersinia), this detection method may further comprise the steps: the test sample that (a) makes the nucleic acid that may contain bacterium to be detected with contact with the nucleic acid molecule more than 2 kinds of at least a portion hybridization of the DnaJ gene of bacteria culture more than 2 kinds to be detected; And b) detect described nucleic acid molecule whether with described test sample in nucleic acid hybridization, wherein, described (a) step is following steps, promptly, use at least a kind of the 1st primer sets with base sequence of a flag sequence and a selectively annealed part to described DnaJ gene, at least a kind of the 2nd primer sets with flag sequence substantially the same with the flag sequence of described the 1st primer sets, implement the polymerase chain reaction of polymerase chain reaction, and implement to utilize described the 1st primer sets the increase at least a portion of described DnaJ gene and the step of the amplified production that described the 1st primer sets obtains that utilizes described the 2nd primer sets to increase.Described polymerase chain reaction step can be implemented single PCR reaction cycle.

The present invention also provides a kind of nucleic acid molecule test kit that is used to detect and identify bacteria culture more than 2 kinds, wherein said more than 2 kinds bacteria culture be selected from streptococcus (Streptococcus), Staphylococcus (Staphylococcus), enterococcus spp (Enterococcus), Peptostreptococcus (PeptoStreptococcus), klebsiella (Klebsiella), Escherichia (Escherichia), enterobacter (Enterobacter), legionella (Legionella), Neisseria (Neisseria), bacillus (Bacillus), Helicobacterium (Helicobacter), Corynebacterium (Corynebacterium), Rhodopseudomonas (Pseudomonas), Bai Huoerde Bacillaceae (Burkholderia), Haemophilus spp (Haemophilus), Bacteroides (Bacteroides), enterococcus spp (Enterococcus), Nuo Kaer Pseudomonas (Nocardia), general Bordetella (Prevotella), Neisseria (Neisseria), Moraxella (Moraxella), Flavobacterium (Flavobacterium), fusobacterium (Clostridium), treponema (Treponema), Sphingobacterium (Sphingobacterium), leptospira (Leptospira), campylobacter (Campylobacter), Listera belongs to (Listeria), the bacterium of Salmonellas (Salmonella) and Yersinia (Yersinia), described test kit comprise the nucleic acid molecule with at least a portion hybridization of the DnaJ gene of described bacterium to be identified.In addition, in this test kit, described nucleic acid molecule comprises at least a kind of the 1st primer sets and at least a kind of the 2nd primer sets with flag sequence substantially the same with the flag sequence of described the 1st primer sets with base sequence of a flag sequence and a selectively annealed part to described DnaJ gene.And the present invention can also adopt the form of the solid phase body that nucleic acid molecule is fixed more than 2 kinds that constitutes such nucleic acid molecule test kit.

Embodiment 1

The streptococcus aureus (S.aureus) of Staphylococcus (Staphylococcus) is to cause the important bacterial classification of poisoning by food, known for 16S rDNA, with the similarity of nearly edge bacterium staphylococcus epidermidis (S.epidermides) be 99.5% (Figure 1A), thereby can not design the specific primer of bacterial classification, but for the DnaJ gene, the similarity of these two bacterial classifications only has 81.1% (Figure 1B).Therefore, can be easily at DnaJ gene design primer.And this primer sequence is guarded (Fig. 2) between the 11 strain streptococcus aureuses (S.aureus) of determining sequence.In the present embodiment, based on the DnaJ sequence of having measured of 9 kinds of pathogen species causing food poisoning, prepare the primer of these pathogenic agent.The primer sequence of each pathogenic agent is as follows.

Streptococcus aureus (Staphylococcus aureus) is used primer

SEQ ID NO：13：5-GGACAAGGATTCAATGGCTCT

SEQ ID NO：14：5-TTGCGGTGCATTTGGATCTCT

Campylobacter jejuni (Campylobacter jejuni) is used primer

SEQ ID NO：67：5-TACTTATAAATGCTCTTGTAAAAC

SEQ ID NO：68：5-CTTTGGACAAGTTTGAAG

Vibrio cholerae (Vibrio cholerae) is used primer

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：44：5-AGCAGCTTATGACCAATACGCC

Vibrio parahaemolyticus (Vibrio parahaemolyticus) is used primer

SEQ ID NO：43：5-CAGGTTTGYTGCACGGCGAAGA

SEQ ID NO：50：5-TGCGAAGAAAGGCTCATCAGAG

Intestinal bacteria (Escherichia coli) are used primer

SEQ ID NO：21：5-TGTTGAGCGCAGCAAAAC

SEQ ID NO：22：5-CAAGACGGATGCGGTCTC

Monocyte hyperplasia Li Site bacterium (Listeria monocytogenes) is used primer

SEQ ID NO：135：GTACAAGCTACATTAGGTGAC

SEQ ID NO：136：GTTCGTTTGAAAATTCCAAGT

(perhaps

SEQ ID NO：137：GGACAACACCTGAAAAATGT

SEQ ID NO：138：AATGGGGTATTTTGTTCCACGT)

Salmonella enteritidis (Salmonella enteritidis) is used primer

SEQ ID NO：139：AAATCAAAAAGGCGTACAAG

SEQ ID NO：140：GAGATTAAAGCAGCCTACGA

Yersinia (Yersinia spp) is used primer

SEQ ID NO：141：CGTTCAGGTACAAGTCAAGG

SEQ ID NO：142：GTGAGGTTCCTATTAACTTTGC

(perhaps

Yersinia entero-colitica (Yersinia enterocolitica) is used primer

SEQ ID NO：143：TACGCGGTGTGACTAAAGAA

SEQ ID NO：144：TATGTACCTGACCTGCACCA)

Clostridium perfringens (Clostridium perfringens) is with primer (wherein, with 16S rDNA as target nucleic acid)

SEQ ID NO：145：GGTGCTTCAGATGATGAG

SEQ ID NO：146：GGAGATAAGGAAGCGGAA

Above-mentioned various primers are mixed in the following manner, and preparation comprises the primer solution A of all primer sets of these 9 kinds bacterial classifications, the PCR reaction soln that refabrication is following.

Primer solution A (each primer 1 μ M): 2 μ l

2 * CYBER pre-mixing Hotstart Ex Taq (Takara): 10 μ l

Streptococcus aureus (S.aureus) DNA (GTC 250 strains): 5ng

It is 20 μ l that adding distil water makes total amount.

Here, in contrast, only use the primer of streptococcus aureus (S.aureus), preparation primer solution B, the PCR reaction soln that refabrication is following.

Primer solution B (each primer 1 μ M): 2 μ l

2 * CYBER pre-mixing Hotstart Ex Taq (Takara): 10 μ l

Streptococcus aureus (S.aureus) DNA (GTC 250 strains): 5ng

It is 20 μ l that adding distil water makes total amount.

In the kapillary of each Light Cycler (Roche Holding Ag), add above-mentioned each PCR reaction soln together with reagent, with Light Cycler to said gene increase (95 ℃: 1 second, 55 ℃: 1 second, 72 ℃: in 10 seconds, carry out 50 circulations).The results are shown in Fig. 3.

Carry out amplification PCR products and carry out amplification PCR products with primer solution B and confirm finally amplification efficiency amplification with much at one with primer solution A.That is, utilize the level of the amplification efficiency that the primer mixture comprise 9 kinds of primer sets and independent primer sets increase identical, show that each primer sets can distinguish the particular sequence of the DnaJ gene of not interfering ground amplification purpose bacterium mutually.

Embodiment 2

The DNA that replaces the streptococcus aureus (S.aureus) of using among the embodiment 1 uses the DNA of following bacterial strain, uses the blended primer sets under same condition.The DNA that increases with the kapillary of Light Cycler (Roche Holding Ag) with the increase of CYBR green fluorescence intensity monitoring.The results are shown in Fig. 4.

[bacterial strain of use]

1. campylobacter jejuni (Campylobacter jejuni) GTC 0259 type strain

2. vibrio cholerae (Vibrio cholerae) GTC 0,037 01 E1 Tol type

3. Vibrio parahaemolyticus (Vibrio parahaemolyticus) GTC 0056 (=CDC A3308)

4. intestinal bacteria (Escherichia coli) GTC 1,061 0157 (VT1 and the VT2 positive)

5. Listeria monocytogenes (Listeria monocytogenes) GTC0149 (=ATCC15313) type strain

6.Salmonella enterica var.Typhimurium GlFU 11566 ST-1

7. yersinia entero-colitica (Yersinia enterocolitica) GTC 0127 type strain

Clostridium perfringens (Clostridium perfringens) GTC 0785 (=ATCC13124)

As shown in Figure 4, confirm that the DNA of each bacterium utilizes the bacterial classification Auele Specific Primer group in the mix primer solution A to increase.Confirmed this each primer sets particular sequence of the DnaJ gene of amplification purpose bacterium with can distinguishing not interference mutually.

Embodiment 3

(amplification of the DnaJ gene of Vibrio (Vibrio) bacterium)

Bacterial classification specific reverse primers beyond following in general forward primer of Vibrio (Vibrio) bacterium (SEQ ID NO:43) and the same genus is mixed and the primer solution that obtains prepares in the following manner.

Primer (SEQ ID NO:43) the 2 μ M that Vibrio (Vibrio) is general

Vibrio cholerae (Vibrio cholerae) (SEQ ID NO:44) 1 μ M

Vibrio mimicus (Vibrio mimicus) (SEQ ID NO:47) 1 μ M

Vibrio vulnificus (Vibrio vulnificus) (SEQ ID NO:53) 1 μ M

Vibrio fluvialis (Vibrio fluvialis) (SEQ ID NO:134) 1 μ M

Vibrio parahaemolyticus (Vibrio parahaemolyticus) (SEQ ID NO:50) 1 μ M

Vibrio alginolyticus (Vibrio alginolyticus) (SEQ ID NO:58) 1 μ M

In addition, this primer solution is prepared the PCR reaction soln in the following manner.

Primer mixing solutions (forward μ M, reverse each 1 μ M): 2 μ l

2 * CYBER pre-mixing Hotstart Ex Taq (Takara): 10 μ l

The DNA of each vibrios (separately): 5ng

Adding water, to make total amount be 20 μ l.

[bacterial strain of employed vibrios]

Vibrio cholerae (Vibrio cholerae) GTC 0,037 01 El Tol type

Vibrio mimicus (Vibrio mimicus) GTC 0334 type strain

Vibrio vulnificus (Vibrio vulnificus) GTC 0116 type strain

Vibrio fluvialis (Vibrio fluvialis) GTC 0315 type strain

Vibrio parahaemolyticus (Vibrio parahaemolyticus) GTC 0056 (=CDC A3308)

Vibrio alginolyticus (Vibrio alginolyticus) GTC 0057 (=ATCC 17449) type strain

In the kapillary of each Light Cycler (Roche Holding Ag) usefulness, add above-mentioned PCR reaction soln together with reagent, with Light Cycler to gene increase (95 ℃: 1 second, 55 ℃: 1 second, 72 ℃: in 10 seconds, carry out 50 and circulate).Confirm the Fig. 5 that the results are shown in of pcr amplification product with agar electrophoresis.

As shown in Figure 5, can obtain each bacterial classification expect the size amplified production.That is, this confirm each primer can be not the particular sequence of the DnaJ gene of amplification purpose bacterium mutually with interfering.

Embodiment 4

(detection sensitivity and the specific amplification of the DnaJ gene amplification in Vibrio (Vibrio) bacterium)

Use the primer solution of preparation among the embodiment 3, compare the detection sensitivity of vibrio cholerae (Vibrio cholerae) GTC 0037 strain with using sample with the ight soil dilution.The bacterium liquid of vibrio cholerae (Vibrio cholerae) GTC 0037 strain is mixed with the ight soil suspension of physiological saline and 10%, extract DNA with phenol-chloroform method.The nucleic acid that serial dilution is extracted, the DNA in the PCR reaction soln of replacement embodiment 3 uses this nucleic acid to implement the PCR reaction, with the sensitivity of electrophoretic method icp gene amplification.The results are shown in Fig. 6.

As shown in Figure 6, quantitatively dilution DNA is measured detection sensitivity, and detection level shown in the figure detects amplified production.In addition, with the DNA of vibrio cholerae with from normal people's ight soil, extract the DNA obtain and mix, mensuration detection sensitivity, its result confirm that detection sensitivity reaches level identical when diluting with distilled water.As known from the above, though each primer sets in this primer solution in the presence of from the inclusion of ight soil, the specific region of the DnaJ gene of each purpose bacterial classification that also can increase specifically, and can the high-sensitivity detection bacterial classification.

Embodiment 5

(amplification and the evaluation of the DnaJ gene of the pathogen species of venereal disease)

The primer that mixing designs based on the specific region of the DnaJ gene of the bacterial classification that uses in the venereal disease diagnosis, the preparation primer solution.

Mycoplasma genitarium primer (SEQ ID NO:71,72)

Chlamydia trachomatis (Chlamydia trachomatis) is used primer (SEQ ID NO:75,76)

Diplococcus gonorrhoeae (Neisseria gonorrhoae) is used primer (SEQ ID NO:79,80)

Here, for following bacterial classification, based on the specific region design primer that is not the gene of DnaJ gene.

Urea mycoplasma (Ureaplasma spp.) primer (Tuf gene)

SEQ ID NO：147：GGATGGTGCAATCTTAGTTATT

SEQ ID NO：148：ACTTGACGCGCTAATAGG

Treponoma palladium (Treponema pallidum) is used primer (16S rDNA gene)

SEQ ID NO：149：TGGGGATAGCCTCTAGAAATAG

SEQ ID NO：150：CCCTTTCCTCTCAAAGACCT

Hsv (Herpes simplex virus) primer (glycoprotein (Glycoprotein) B protein gene)

SEQ ID NO：151：CTGGTCAGCTTTCGGTACG

SEQ ID NO：152：CCGCAGCTCGTTGTTCTC

Papilloma virus (Papilloma virus) primer (albumen E6 gene)

SEQ ID NO：153：TCAAAAGCCACTGTGTCCT

SEQ ID NO：154：CGACCCCTTATATTATGGAATCTT

Candida albicans (Candida albicans) primer (26S rDNA gene)

SEQ ID NO：155：TCAGTAGCGGCGAGTGAA

SEQ ID NO：156：GCCCAAAGATACCTTCTTCAAAT

Trichomonas vaginalis (Trichomonas vaginalis) is used primer (18S rDNA gene)

SEQ ID NO：157：GAAATCATAGTTCTTGGGCTCTG

SEQ ID NO：158：CCTTCCGTCAATTCCTTCAAG

Above-mentioned each primer is mixed preparation PCR reaction soln by following main points.

Primer solution (each primer 1 μ M): 2 μ l

2 * CYBER pre-mixing Hotstart Ex Taq (Takara): 10 μ l

The DNA of each bacterial strain: 5ng

Adding water, to make total amount be 20 μ l.

In the kapillary of each Light Cycler (Roche Holding Ag) usefulness, add above-mentioned PCR solution together with reagent, carry out gene amplification (95 ℃: 1 second, 55 ℃: 1 second, 72 ℃: in 10 seconds, carry out 50 circulations) with Light Cycler.Use following bacterial strain in this experiment.The DNA that increases in the kapillary of Light Cycler (Roche Holding Ag) is monitored in increase by the CYBR green fluorescence intensity, the results are shown in Fig. 7.

[employed bacterial strain]

1Mycoplasma genitarium GGS 2289 (from the clone of clinical sample)

2 chlamydia trachomatises (Chlamydia trachomatis) GGS 922 (from the clone of clinical sample)

3 Diplococcus gonorrhoeaes (Neisseria gonorrhoae) GTC 02085 (=ATCC49226)

4, urea mycoplasma (Ureaplasma spp.) GGS 1699 (from the clone of clinical sample)

5 Treponoma palladiums (Treponema pallidum) GGS 745 (from the clone of clinical sample)

6 hsvs (Herpes simplex virus) GGS 1943 (from the clone of clinical sample)

7 papilloma viruss (Papilloma virus) GGS 1944 (from the clone of clinical sample)

8 Candida albicanss (Candida albicans) GTC 00654 (=IFM 5801)

9 Trichomonas vaginalises (Trichomonas vaginalis) GGS 1941 (from the clone of clinical sample)

GTC: the abundant typical culture collection of qi center

GGS: the abundant recombinant strain preservation of qi center

ATCC: American type culture collection, the U.S.

NCTC: Britain country typical culture collection center, Britain

IFM: pathomycete and microbial toxin disease research centre, Chiba University)

As shown in Figure 7, the increased DNA of each bacterial strain.That is, as can be known this each primer can be not the particular sequence of the DnaJ gene of amplification purpose bacterium mutually with interfering.

Embodiment 6

(amplification and the evaluation of the DnaJ gene of the pathogenic agent bacterial classification of pneumonia and pharyngitis)

The primer that will design based on the specific region of the DnaJ gene of the pathogenic agent bacterial classification that causes pneumonia and pharyngitis mixes the preparation primer solution.

Streptococcus pneumoniae (Streptococcus pneumoniae) is used primer (SEQ ID NO:9,10)

Streptococcus pyogenes (Streptococcus pyogenes) primer (SEQ ID NO:1,2)

Streptococcus aureus (Staphylococcus aureus) is used primer (SEQ ID NO:13,14)

Klebsiella pneumoniae (Klebsiella pneumoniae) primer (SEQ ID NO:17,18)

Intestinal bacteria (Escherichia coli) are used primer (SEQ ID NO:21,22)

Mycobacterium tuberculosis (Mycobacterium tuberculosis) primer (SEQ ID NO:25,26)

Chlamydia pneumoniae (Chlamydia pneumoniae) primer (SEQ ID NO:39,40)

Legionella pneumophilia (Legionella pneumophila) is used primer (SEQ ID NO:35,36)

Mycoplasma pneumoniae (Mycoplasma pneumoniae) primer (SEQ ID NO:31,32)

Mycobacterium (Mycobacterium) group primer (SEQ ID NO:29,30)

[employed bacterial strain]

Streptococcus pneumoniae (Streptococcus pneumoniae) GTC 01147 (=NCTC 7465)

Streptococcus pyogenes (Streptococcus pyogenes) GTC 01839

Streptococcus aureus (Staphylococcus aureus) GTC 02805

Klebsiella pneumoniae (Klebsiella pneumoniae) GTC 00107 (=NCTC9633)

Intestinal bacteria (Escherichia coli) GTC 09975 (=ATCC 25868)

Mycobacterium tuberculosis (Mycobacterium tuberculosis) GTC 12850 (=ATCC27294)

Chlamydia pneumoniae (Chlamydia pneumoniae) ATCC VR 145

Legionella pneumophilia (Legionella pneumophila) GTC 00702 (=ATCC 33737)

Mycoplasma pneumoniae (Mycoplasma pneumoniae) GTC 00666 (ATCC 15531)

Mycobacterium avium (Mycobacterium avium) GTC 00603 (=ATCC 25291)

GTC: the abundant typical culture collection of qi center

GGS: the abundant recombinant strain preservation of qi center

ATCC: American type culture collection, the U.S.

NCTC: Britain country typical culture collection center, Britain

IFM: pathomycete and microbial toxin disease research centre, Chiba University)

Above-mentioned each primer is mixed preparation PCR reaction soln by following main points.

Primer solution (each primer 1 μ M): 2 μ l

2 * CYBER pre-mixing Hotstart Ex Taq (Takara): 10 μ l

The DNA of each bacterial strain: 5ng

Adding water, to make total amount be 20 μ l.

In the kapillary of each Light Cycler (Roche Holding Ag) usefulness, add reagent, above-mentioned PCR reaction soln is carried out gene amplification (95 ℃: 1 second, 55 ℃: 1 second, 72 ℃: in 10 seconds, carry out 50 circulations) with LightCycler.Use above-mentioned bacterial strains DNA in this experiment.Confirm pcr amplification product with agar electrophoresis, its result, as can be known, these primers are to the specific region of the DnaJ gene that can distinguish the purpose bacterial classification that increases specifically, and can detect bacterial classification in high sensitivity.

Embodiment 7

(amplification of the DnaJ gene of the bacterial classification of Mycobacterium (Mycobacterium) bacterium and the evaluation of being undertaken by order-checking)

With Primer working method checked order (being shown in Fig. 8) in the almost whole zone of the DnaJ gene of the type strain of 3 subspecies of 55 bacterial classifications of Mycobacterium (Mycobacterium) bacterium, and will utilize the extension increasing sequence of the 386bp that SEQ ID NO:29,30,131,132 and 133 obtains and almost whole zone 188bp the two come comparative sequences.Its result and the existing gene RpoB that has analyzed are compared.The results are shown in Fig. 9 and 10.

As shown in Figures 9 and 10, be 96.6% for 16S rDNA sequence average similarity, dnaJ gene order average similarity is 80.7%, compare the similarity of the analog value 90.0～90.7% of rpoB, hsp65, polymorphism is the highest.

In addition, the similarity of the 16SrDNA sequence of Kan Sasishi mycobacterium-mycobacterium gastri (M.kansasii-M.gastri) is 100%, can not distinguish come (Figure 11), when comparing the DnaJ gene, Kan Sasishi mycobacterium-mycobacterium gastri (M.kansasii-M.gastri) has 95.5% similarity.Therefore, based on biochemical characteristic, clinical separation strain (KPM strain) with above-mentioned SEQ ID NO:29,30,131,132 and 133 amplification 23 Kan Sasishi mycobacteriums (M.kansasii) (20 strain) and mycobacterium gastri (M.gastri) (2 strain), use the sequence of all type strains that checked order, manufacturing system is set.Its result, as shown in figure 12, all strains with coming to the same thing that biochemical characteristic is identified.

In addition, because mycobacterium avium (M.avium) is similar to the biochemical characteristic of Mycobacterium intracellulare (M.intracellulare), be difficult to identification, therefore, similarly measure sequence with the 16 strain clinical separation strains (the KPM strain among the figure) that are referred to as mycobacterium avium-Mycobacterium intracellulare (M.avium-M.intracellulae) group (MAC group) and compare.It the results are shown in Figure 13.

As shown in figure 13,9 strains are mycobacterium avium (M.avium), and 3 strains are Mycobacterium intracellulare (M.intracellulare), and M.chimaera, 1 strain that 3 strains are accredited as new classification are accredited as lymphoid tuberculosis mycobacterium (M.scrofulaceum).As known from the above, the method for identifying with these whole base sequences and partial sequence is effective for the correct evaluation of the flora that can not discern with biochemical characteristic.

Embodiment 8

In the present embodiment, as target nucleic acid, implement multiplex PCR with legionella pneumophilia (Legionella pneumophila) GTC1279DNA.As primer, prepare the mixture (containing each ultimate density of forward primer (SEQ ID NO:37) and reverse primer (SEQ ID NO:38) (all only being label) is 0.1 μ M) of the amplimer group of 18 groups of mixtures (forward primer (SEQ ID NO:1～18) and each ultimate density of reverse primer (SEQID NO:19～36) (all having mark) are 0.1 μ M) that have an annealing primer of mark sequence identical with having each mark of having with these annealing primer.The sequence of these primers is shown in Figure 15.

Use these primer mixed solutions and dna solution (legionella pneumophilia (Legionellapneumophila) GTC 1279DNA 1 μ g/ml), prepare the PCR reaction soln according to following composition, use Light Cycler (Roche Holding Ag), implement the PCR reaction according to following condition.As a comparative example, preparation contain with 5 ' end from the forward primer of above-mentioned 18 kinds of primer sets remove 18 bases flag sequence annealing primer and removed the mixed solution (each ultimate density of forward primer and reverse primer (all not having label) is 0.1 μ M) of the annealing primer that does not have label of annealing primer of the flag sequence of 27 bases from 5 ' end of reverse primer, prepare the PCR reaction soln according to following composition, carry out PCR with the same operation of embodiment.The amplification monitoring result is shown in Figure 16.

(composition of the PCR reaction soln of embodiment 8)

2 * SYBR pre-mixing Ex Taq (Takara): 10 μ l

Primer mixed solution (forward primer, each 1 μ M of reverse primer): 2 μ l

Amplimer mixed solution (forward primer, each 10 μ M of reverse primer): 2 μ l

Legionella pneumophilia (Legionella pneumophila) GTC 1279DNA (1ng/ml): 1 μ l

dH ₂O ：5μl

Total amount: 20 μ l

(composition of the PCR reaction soln of comparative example)

2 * SYBR pre-mixing Ex Taq (Takara): 10 μ l

Primer mixed solution (forward primer, each 1 μ M of reverse primer): 2 μ l

Legionella pneumophilia (Legionella pneumophila) GTC 1279DNA (1ng/ml): 1 μ l

dH ₂O ：7μl

Total amount: 20 μ l

(reaction conditions)

95 ℃: 1 second, 60 ℃: 1 second, 72 ℃: 60 circulations in 5 seconds

As shown in figure 16, utilize the primer sets and the amplimer group of the tape label of embodiment 8, for legionella pneumophilia (Legionella pneumophila) GTC 1279DNA, begin to increase at about 35 circulation back amplified signals, approximately show good detection sensitivity after the circulation 40.With respect to this, in not using the comparative example of amplimer, even after 50 circulations, target nucleic acid does not increase fully yet.As known from the above, the annealing primer and flag sequence and amplification primers that have selectively annealed anneal sequence to target nucleic acid by use with flag sequence identical with the flag sequence of this annealing primer, even use multiple primer sets, specific target nucleic acid fully also can increase.

Embodiment 9

In the present embodiment, the annealing primer that makes among the embodiment 8 tape label of making and amplimer cooperate that ratio is as shown in table 2 to be changed in 1: 1～1: 50, when preparing the PCR reaction soln as shown below according to following condition enforcement PCR.Here, as target nucleic acid, use mycoplasma pneumoniae (Mycoplasma pneumoniae) GTC 2987DNA.As a comparative example, use the independent primer solution (4 μ m, 8 μ M) of prior art, prepare the PCR reaction soln, under the condition identical, implement PCR with embodiment according to following composition.The results are shown in Figure 17.Here, independent primer is the sequence of removing from the forward primer of mycoplasma pneumoniae (Mycoplasma pneumoniae) 16S rDNA and reverse primer behind the flag sequence.

Table 2

The primer mixture mixture ratio

No.1 0.5 μ M: 2 μ l amplification labels (0.5 μ M), the 2 μ l 1: 1 that anneal

No.2 0.5 μ M: 2 μ l amplification labels (2.5 μ M), the 2 μ l 1: 5 that anneal

No.3 0.5 μ M: 2 μ l amplification labels (5 μ M), the 2 μ l 1: 10 that anneal

No.4 0.5 μ M: 2 μ l amplification labels (10 μ M), the 2 μ l 1: 20 that anneal

No.5 0.5 μ M: 2 μ l amplification labels (20 μ M), the 2 μ l 1: 40 that anneal

No.6 0.5 μ M: 2 μ l amplification labels (25 μ M), the 2 μ l 1: 50 that anneal

(composition of the PCR reaction soln of embodiment 9)

2 * SYBR pre-mixing Hot Start (Takara): 10 μ l

Mycoplasma pneumoniae (Mycoplasma pneumoniae) GTC 2987DNA (1ng/ml): 1 μ l

Primer mixed solution: 4 μ l

dH ₂O ：5μl

Total amount: 20 μ l

(composition of the PCR reaction soln of comparative example)

2 * SYBR pre-mixing Hot Start (Takara): 10 μ l

Mycoplasma pneumoniae (Mycoplasma pneumoniae) GTC 2987DNA (1ng/ml): 1 μ l

Independent primer * (4 μ M, 8 μ M) mixed solution: 4 μ l

dH ₂O ：5μl

Total amount: 20 μ l

(reaction conditions)

95 ℃: 1 second, 60 ℃: 1 second, 72 ℃: 60 circulations in 5 seconds

As shown in figure 17, be accompanied by the rising of the ratio of amplimer and annealing primer, amplification efficiency increases, and when this ratio is more than 10 times the time, shows amplification efficiency identical when increasing with the independent primer that uses 0.4 μ M concentration.

Embodiment 10

In the present embodiment, the designated area of the gene that is had with bacterial disease pathogenic microorganism and viral pathogens with Figure 18 and diarrhoea shown in Figure 19 is set at 1: 50 mode as the annealing primer of target nucleic acid and the mixture ratio of amplimer, prepare the PCR reaction soln according to following composition, under following reaction conditions, implement PCR simultaneously.After implementing PCR, PCR product and the silicon chip (Japan's steel plate system) that is mounted with probe shown in Figure 20 were hybridized 30 minutes down at 55 ℃.After the hybridization, washing silicon wafer is with the fluorescence intensity of laser scanning Scan Array 4000 (GSI Lumonics society systems) mensuration Cye5.The results are shown in Figure 21.Here, Figure 21 has also expressed the positional information of the probe of revealing.

(composition of the PCR reaction soln of embodiment 10)

2 * SYBR pre-mixing Hot Start (Takara): 10 μ l

Vibrio cholerae (Vibrio cholerae) DNA (1ng/ml): 1 μ l

Adenovirus (Adenovirus) DNA (1ng/ml): 1 μ l

Annealing primer mixed solution (each 0.2 μ M): 2 μ l

Amplimer mixed solution (each 10 μ M): 2 μ l

dH ₂O ：4μl

Total amount: 20 μ l

(reaction conditions)

95 ℃: 1 second, 60 ℃: 1 second, 72 ℃: 60 circulations in 5 seconds

As shown in figure 21, even comprise 49 kinds of annealing primer groups, also can distinguish the good and sensitivity of specificity and detect 2 kinds of target nucleic acids well.

Embodiment 11

In the present embodiment, prepare 16 kinds of venereal diseases (STD) shown in Figure 22 mixture (each primer ultimate density 0.02 μ M) and amplimer group mixed solution (each 10 μ M) with the annealing primer group, as shown below, it with the ratio of amplimer and annealing primer 20 mode, prepare the PCR reaction soln by following composition, implement PCR according to following condition.Behind the amplified reaction, the fluorescent bead from the Luminax company that is combined with probe shown in Figure 23 to the PCR product and the streptavidin-phycoerythrin (Streptavidin-phycoerythrin) that add, and at room temperature reacted 30 minutes, with the Luminax 100 fluorometric assay systems measurement fluorescence intensities of Luminax company.The results are shown in Figure 24.

As shown in figure 24, contained unique target nucleic acid combines with high specific with the probe of Chlamydiatoracomatis in the PCR reaction soln.As known from the above, in the PCR product, this target nucleic acid increases specifically.

Embodiment 12

In the present embodiment, prepare 4 kinds of primer mixtures shown in Figure 25.Each primer shown in Figure 25 and that use in the present embodiment is the primer with sequence of having mentioned.Make each 200pg/ml of DNA, each the annealing primer 0.075 μ M, each amplimer 0.8 μ M, the 16S rDNA universal primer 0.2 μ M that comprise each bacterial classification shown in Figure 25 in the primer PCR reaction soln.Here, archaeal dna polymerase uses SYBR pre-mixing Hot Start (TaKaRa).For this PCR reaction soln, heating was carried out 95 ℃: 1 second, 55 ℃: 1 second, 72 ℃: the circulation in 10 seconds 50 times after 1 minute under each circulated in 95 ℃.Result's (CTm value) is shown in Figure 26.Here, the CTm value is the PCR cycle number when reaching the signals such as fluorescence intensity of half peak value that becomes amplification curve when carrying out a subdifferential.We can say that the CTm value is more little, amplification efficiency is good more.

As shown in figure 26, with respect to the CTm value is about 40～46 as target nucleic acid, when using the primer mixture A that comprises annealing primer and amplimer to implement PCR with 16S rDNA gene, when implementing PCR as the primer mixture B of reverse primer, observe the tendency that the CTm value has reduction in order to the universal sequence of 16S rRNA.That is, when designing annealing primer with 16S rRNA as target nucleic acid, even mix the primer that different bacterial classifications takes place in 8 kinds of systems, amplification efficiency is compared the tendency that also has reduction on the contrary with reference examples.Therefore, 16S rRNA is the target nucleic acid that is not suitable as multiplex PCR of the present invention as can be known.

With respect to this, result by mix primer C and D can be clear and definite, be target nucleic acid with the DnaJ gene, when using annealing primer and amplimer, even there are a plurality of other primers (totally 18 kinds), also can be specifically with primer annealing to target sequence and amplification effectively.Therefore, the DnaJ gene is useful as the target nucleic acid of such multiplex PCR as can be known.

Sequence table

SEQ ID NO:1～86,131,132,133,135～158,159～294 and 326～357: primer

SEQ ID NO:87～130,134,295～325 and 358～367: probe

Sequence table

＜110〉Univ Gifu (Gifu University)

AMR Co., Ltd. (AMR, Inc.)

＜120〉utilize the Bacteria Detection and application (the Detection Of Bacterium By Utilizing DnaJ and thereof of DnaJ gene

use Thereof)

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gatcaatttg gtcatgctgg t 21

<210>62

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>62

gaagccacca ccgaagtc 18

<210>63

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>63

aaagcgtatc gtcgtttgg 19

<210>64

<211>24

<212>DNA

<213>Artificial

<220>

<223>primer

<400>64

atcatcactt aacacttcat atgc 24

<210>65

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>65

cgcagtacga tcaatttgg 19

<210>66

<211>25

<212>DNA

<213>Artificial

<220>

<223>primer

<400>66

caaagaaaga actaaatata tcttc 25

<210>67

<211>24

<212>DNA

<213>Artificial

<220>

<223>primer

<400>67

tacttataaa tgctcttgta aaac 24

<210>68

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>68

ctttggacaa gtttgaag 18

<210>69

<211>25

<212>DNA

<213>Artificial

<220>

<223>primer

<400>69

tagactttac ttataaatgc tcttg 25

<210>70

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>70

gacactttgg acaagtttg 19

<210>71

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>71

gaagggttaa atgcttctgg 20

<210>72

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>72

caccatccat tccaaagg 18

<210>73

<211>25

<212>DNA

<213>Artificial

<220>

<223>primer

<400>73

aagggattat tatgaagttc taggg 25

<210>74

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>74

cattttctgc tttatgacga tc 22

<210>75

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>75

gggatttcct tcttgatcc 19

<210>76

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>76

catggggatg atttagtttt ag 22

<210>77

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>77

aaagaaaccg cctccatta 19

<210>78

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>78

gcatggggaa tatggaagac 20

<210>79

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>79

ggcggggagt tagaagtg 18

<210>80

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>80

gacaccctta cctttcacg 19

<210>81

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>81

cgggcaaaca tattaaagaa cc 22

<210>82

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>82

cgggaatatt gacttccac 19

<210>83

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>83

ttggaagtgc cgaccttg 18

<210>84

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>84

gatttgacac ccttaccctt cg 22

<210>85

<211>21

<212>DNA

<213>Artificial

<220>

<223>primer

<400>85

gggcaaacac attaaagaac c 21

<210>86

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>86

gcgggaatat tgacttcca 19

<210>87

<211>28

<212>DNA

<213>Artificial

<220>

<223>probe

<400>87

ttaccatcca gacgtaagta aagaagaa 28

<210>88

<211>24

<212>DNA

<213>Artificial

<220>

<223>probe

<400>88

ttagccaccg ccgaagtctc ctcc 24

<210>89

<211>25

<212>DNA

<213>Artificial

<220>

<223>probe

<400>89

ttcgccgtat tgatcataag cagca 25

<210>90

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>90

ttcctgagcc taaacaagtg ccg 23

<210>91

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>91

ttaaaaccgc catttgcccc ag 22

<210>92

<211>28

<212>DNA

<213>Artificial

<220>

<223>probe

<400>92

tcttcctgaa caagtatgac atgatgac 28

<210>93

<211>24

<212>DNA

<213>Artificial

<220>

<223>probe

<400>93

ttcgtgctgc ctatgaccag tatg 24

<210>94

<211>27

<212>DNA

<213>Artificial

<220>

<223>probe

<400>94

tcgatgatac ttaacttcct tctcagt 27

<210>95

<211>24

<212>DNA

<213>Artificial

<220>

<223>probe

<400>95

tacagtctca ccctcaccct tatg 24

<210>96

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>96

ttcacaggca ctacaggtca cc 22

<210>97

<211>26

<212>DNA

<213>Artificial

<220>

<223>probe

<400>97

ttcaatccgt aaagatgtaa catgcg 26

<210>98

<211>28

<212>DNA

<213>Artificial

<220>

<223>probe

<400>98

ttcaatccgt aaagatgtaa catgcgaa 28

<210>99

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>99

ttcgtacaga tcgcctgccg 20

<210>100

<211>24

<212>DNA

<213>Artificial

<220>

<223>probe

<400>100

cgcgccggca ggcgatctgt acgt 24

<210>101

<211>27

<212>DNA

<213>Artificial

<220>

<223>probe

<400>101

aagagatccg cattccgact ctggaag 27

<210>102

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>102

ttaagagatc cgcattccga ct 22

<210>103

<211>21

<212>DNA

<213>Artificial

<220>

<223>probe

<400>103

tcggggtcgg tggagacggc g 21

<210>104

<211>26

<212>DNA

<213>Artificial

<220>

<223>probe

<400>104

ttctcctctg atgccagtcc tgaaga 26

<210>105

<211>27

<212>DNA

<213>Artificial

<220>

<223>probe

<400>105

tagcagggtt taatcctttt gacatct 27

<210>106

<211>25

<212>DNA

<213>Artificial

<220>

<223>probe

<400>106

taaacgctag ttctcaagac ataaa 25

<210>107

<211>24

<212>DNA

<213>Artificial

<220>

<223>probe

<400>107

ttcgcaaggc atcttccatg ttcc 24

<210>108

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>108

tttcttactg gctccttgac ga 22

<210>109

<211>26

<212>DNA

<213>Artificial

<220>

<223>probe

<400>109

ttagccgcat caacaaatcc aatagg 26

<210>110

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>110

ttaccgaaat cgccgccaaa 20

<210>111

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>111

ttcttgaccg ccttattccg cc 22

<210>112

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>112

tgccgtggcg cggggcggaa ta 22

<210>113

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>113

ttcttgaccg ccttattccg cc 22

<210>114

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>114

tgccgtggcg tggggcggaa ta 22

<210>115

<211>28

<212>DNA

<213>Artificial

<220>

<223>probe

<400>115

taagaagttg aaattaccgt tccaagac 28

<210>116

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>116

aaagaagttg aaattaccgt tcc 23

<210>117

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>117

ttccagcgtc agttccatgt tg 22

<210>118

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>118

tcgagcagcc gcgtaccgct tct 23

<210>119

<211>22

<212>DNA

<213>Artificial

<220>

<223>probe

<400>119

tccatattga tcgtaagccg ct 22

<210>120

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>120

tcgagacacc acgaacggcc tct 23

<210>121

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>121

ttaatcagca ccgccgccac 20

<210>122

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>122

tcgttacacc acgaaccgct tct 23

<210>123

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>123

ttcagcaccg cctccaccaa 20

<210>124

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>124

tagttacacc acgaacggct tct 23

<210>125

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>125

ttcgaagccg ccgccaccaa 20

<210>126

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>126

tcgaacatcc acgaaccgct tct 23

<210>127

<211>20

<212>DNA

<213>Artificial

<220>

<223>probe

<400>127

ttaatcagca ccgccaccgc 20

<210>128

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>128

tcgagcagcc gcgaaccgct tct 23

<210>129

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>129

tgtaatggaa caggggcta 19

<210>130

<211>23

<212>DNA

<213>Artificial

<220>

<223>probe

<400>130

tccatcttta gcccctgttc cat 23

<210>131

<211>21

<212>DNA

<213>Artificial

<220>

<223>primer

<400>131

gtgartgggt cgaraargac t 21

<210>132

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<220>

<221>misc_feature

<222>(3)..(3)

<223>inosine

<400>132

cgngtytcgt cgtaytcctt 20

<210>133

<211>17

<212>DNA

<213>Artificial

<220>

<223>primer

<400>133

cagcgrttac cygccca 17

<210>134

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>134

gtacgaaatt ctgaccgatc aa 22

<210>135

<211>21

<212>DNA

<213>Artificial

<220>

<223>primer

<400>135

gtacaagcta cattaggtga c 21

<210>136

<211>21

<212>DNA

<213>Artificial

<220>

<223>primer

<400>136

gttcgtttga aaattccaag t 21

<210>137

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>137

ggacaacacc tgaaaaatgt 20

<210>138

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>138

aatggggtat tttgttccac gt 22

<210>139

<211>20

<212>DNA

<213>Artificial

<220>

<223>pr imer

<400>139

aaatcaaaaa ggcgtacaag 20

<210>140

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>140

gagattaaag cagcctacga 20

<210>141

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>141

cgttcaggta caagtcaagg 20

<210>142

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>142

gtgaggttcc tattaacttt gc 22

<210>143

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>143

tacgcggtgt gactaaagaa 20

<210>144

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>144

tatgtacctg acctgcacca 20

<210>145

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>145

ggtgcttcag atgatgag 18

<210>146

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>146

ggagataagg aagcggaa 18

<210>147

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>147

ggatggtgca atcttagtta tt 22

<210>148

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>148

acttgacgcg ctaatagg 18

<210>149

<211>22

<212>DNA

<213>Artificial

<220>

<223>primer

<400>149

tggggatagc ctctagaaat ag 22

<210>150

<211>20

<212>DNA

<213>Artificial

<220>

<223>primer

<400>150

ccctttcctc tcaaagacct 20

<210>151

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>151

ctggtcagct ttcggtacg 19

<210>152

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>152

ccgcagctcg ttgttctc 18

<210>153

<211>19

<212>DNA

<213>Artificial

<220>

<223>primer

<400>153

tcaaaagcca ctgtgtcct 19

<210>154

<211>24

<212>DNA

<213>Artificial

<220>

<223>primer

<400>154

cgacccctta tattatggaa tctt 24

<210>155

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>155

tcagtagcgg cgagtgaa 18

<210>156

<211>23

<212>DNA

<213>Artificial

<220>

<223>primer

<400>156

gcccaaagat accttcttca aat 23

<210>157

<211>23

<212>DNA

<213>Artificial

<220>

<223>primer

<400>157

gaaatcatag ttcttgggct ctg 23

<210>158

<211>21

<212>DNA

<213>Artificial

<220>

<223>primer

<400>158

ccttccgtca attccttcaa g 21

<210>159

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>159

tagcaggatc cctctaagat agataatctg ccctcaactt g 41

<210>160

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>160

tagcaggatc cctctaagct aataccgcat aatctctttg g 41

<210>161

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>161

tagcaggatc cctctaaggg gataacgacg ggaaactg 38

<210>162

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>162

tagcaggatc cctctaagga caacttgggg aaactcaag 39

<210>163

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>163

tagcaggatc cctctaaggg gaaactgggt ctaatacc 38

<210>164

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>164

tagcaggatc cctctaagct agctaatacc gcataagaac 40

<210>165

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>165

tagcaggatc cctctaagtc agtacggaat aacatttag 39

<210>166

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>166

tagcaggatc cctctaagga acttgcctgg tagtaggg 38

<210>167

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>167

tagcaggatc cctctaagtc agtacggaat aacttttag 39

<210>168

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>168

tagcaggatc cctctaagtc tacctagtag tatgggatag c 41

<210>169

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>169

tagcaggatc cctctaagat tccaggaatg cagaaagacc 40

<210>170

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>170

tagcaggatc cctctaagtt gttcggtggc ttgttc 36

<210>171

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>171

tagcaggatc cctctaagtg ttgagcgcag caaaac 36

<210>172

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>172

tagcaggatc cctctaagcc ctgtcggtta aaattcc 37

<210>173

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>173

tagcaggatc cctctaagca ggtgcagatg cgtcag 36

<210>174

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>174

tagcaggatc cctctaagag ttaggaagct tgcttctg 38

<210>175

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>175

tagcaggatc cctctaagac gtcctgaggg agaaagtgg 39

<210>176

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>176

tagcaggatc cctctaagac cgcataagag agactaacg 39

<210>177

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>177

aattctaata cgactcacta tagggagtaa actcttcctc aaccgaaag 49

<210>178

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>178

aattctaata cgactcacta tagggagcca ccaacaagct aatccg 46

<210>179

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>179

aattctaata cgactcacta tagggagggc tcatctcatg gcatgcg 47

<210>180

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>180

aattctaata cgactcacta tagggagacg caggctaatc ttaaagcg 48

<210>181

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>181

aattctaata cgactcacta tagggagacc ccaccaacaa gctgatagg 49

<210>182

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>182

aattctaata cgactcacta tagggagcca ccaactagct gatatgg 47

<210>183

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>183

aattctaata cgactcacta tagggagaag ccattacctt accaac 46

<210>184

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>184

aattctaata cgactcacta tagggagcac caactagcta atctaacg 48

<210>185

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>185

aattctaata cgactcacta tagggagtca tccatcagca ataaatc 47

<210>186

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>186

aattctaata cgactcacta tagggagcat ctaatagcga taaatctttc c 51

<210>187

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>187

aattctaata cgactcacta tagggagctc atatgctgtt cctgtattg 49

<210>188

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>188

aattctaata cgactcacta tagggagcct ccacgaaatc caactc 46

<210>189

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>189

aattctaata cgactcacta tagggagcaa gacggatgcg gtctc 45

<210>190

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>190

aattctaata cgactcacta tagggagctc aaagatagcg tgctg 45

<210>191

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>191

aattctaata cgactcacta tagggagcgg atctttgatg atctgacc 48

<210>192

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>192

aattctaata cgactcacta tagggagggg tttccccaag ttatcc 46

<210>193

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>193

aattctaata cgactcacta tagggagctt taccccacca actagc 46

<210>194

<211>52

<212>DNA

<213>Artificial

<220>

<223>primer

<400>194

aattctaata cgactcacta tagggaggag tggttgtttg gttggttatt cg 52

<210>195

<211>18

<212>DNA

<213>Artificial

<220>

<223>primer

<400>195

tagcaggatc cctctaag 18

<210>196

<211>27

<212>DNA

<213>Artificial

<220>

<223>primer

<400>196

aattctaata cgactcacta tagggag 27

<210>197

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>197

tagcaggatc cctctaaggg ataacagttg gaaacgac 38

<210>198

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>198

tagcaggatc cctctaaggg ataacagttg gaaacgac 38

<210>199

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>199

tagcaggatc cctctaagtt gctccctgtg attagtggc 39

<210>200

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>200

tagcaggatc cctctaaggc accaggaatg tcttgtag 38

<210>201

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>201

tagcaggatc cctctaagca ggtttgytgc acggcgaaga 40

<210>202

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>202

tagcaggatc cctctaagca ggtttgytgc acggcgaaga 40

<210>203

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>203

tagcaggatc cctctaagga aaagatwtgt caatytccac tt 42

<210>204

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>204

tagcaggatc cctctaagaa gaaatcaatt gtgaagttat tg 42

<210>205

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>205

tagcaggatc cctctaaggt aagagcagca agatcactat ta 42

<210>206

<211>43

<212>DNA

<213>Artificial

<220>

<223>primer

<400>206

tagcaggatc cctctaagca aaatattgrc ttccactgag ctt 43

<210>207

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>207

tagcaggatc cctctaagtg aacagraggt ttctgcgtta 40

<210>208

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>208

tagcaggatc cctctaagaa tttaccttag atttctcgac ag 42

<210>209

<211>43

<212>DNA

<213>Artificial

<220>

<223>primer

<400>209

tagcaggatc cctctaagtt taataatata tcagcgatac tgg 43

<210>210

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>210

tagcaggatc cctctaagat caaaccaagg ccagtatta 39

<210>211

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>211

tagcaggatc cctctaagct ttctgtattg tctttttcac c 41

<210>212

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>212

tagcaggatc cctctaagca tcaacacagt atatccgaag 40

<210>213

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>213

tagcaggatc cctctaagct tattaaaaat tccaatacaa gc 42

<210>214

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>214

tagcaggatc cctctaagtc aatggaaaga tgtaaatagt ga 42

<210>215

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>215

tagcaggatc cctctaagca aaaagaatca gtayttttca ga 42

<210>216

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>216

tagcaggatc cctctaagtt ttctttatgc atgttcttct aa 42

<210>217

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>217

tagcaggatc cctctaagct caatactttt caccttaatc g 41

<210>218

<211>42

<212>DNA

<213>Artificial

<220>

<223>primer

<400>218

tagcaggatc cctctaagaa gtatcaggga tgtcctataa ag 42

<210>219

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>219

aattctaata cgactcacta tagggagagc cattaccyca ccaacta 47

<210>220

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>220

aattctaata cgactcacta tagggagagc cattaccyca ccaacta 47

<210>221

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>221

aattctaata cgactcacta tagggagaaa ttctttccac ccacag 46

<210>222

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>222

aattctaata cgactcacta tagggagggt tcacctacgg atacct 46

<210>223

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>223

aattctaata cgactcacta tagggagaga agcggttcgc ggctgctcg 49

<210>224

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>224

aattctaata cgactcacta tagggaggaa gccgttcgtg gtgtaact 48

<210>225

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>225

aattctaata cgactcacta tagggaggrt cgtcataacc ttctgctt 48

<210>226

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>226

aattctaata cgactcacta tagggagttt cctactcctc ctttactttt a 51

<210>227

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>227

aattctaata cgactcacta tagggagctt gtatttctca gcatcattaa c 51

<210>228

<211>52

<212>DNA

<213>Artificial

<220>

<223>primer

<400>228

aattctaata cgactcacta tagggagagc gttagttggg aacttgcatt ta 52

<210>229

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>229

aattctaata cgactcacta tagggagaac cacactaact acttgctaa 49

<210>230

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>230

aattctaata cgactcacta tagggagctc ctgatgaaat agtctgtaat g 51

<210>231

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>231

aattctaata cgactcacta tagggagcag ttatctgaca ttctggttg 49

<210>232

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>232

aattctaata cgactcacta tagggagctt catcactctg acagtatagg t 51

<210>233

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>233

aattctaata cgactcacta tagggagagc aggattacaa cacaatt 47

<210>234

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>234

aattctaata cgactcacta tagggagttt gtagtccttc catgacac 48

<210>235

<211>50

<212>DNA

<213>Artificial

<220>

<223>primer

<400>235

aattctaata cgactcacta tagggagtgt tttatgtatt caagagcaat 50

<210>236

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>236

aattctaata cgactcacta tagggaggta tcatttgatg ctgattctat t 51

<210>237

<211>50

<212>DNA

<213>Artificial

<220>

<223>primer

<400>237

aattctaata cgactcacta tagggagaaa gctctctacg gaagaatcta 50

<210>238

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>238

aattctaata cgactcacta tagggaggtt caagtcctag ttttaaagga t 51

<210>239

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>239

aattctaata cgactcacta tagggaggat ttttacgcct tattttacga c 51

<210>240

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>240

aattctaata cgactcacta tagggagctt tataggacat ccctgatact t 51

<210>241

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<220>

<221>misc_feature

<222>(30)..(30)

<223>n is a，c，g，or t

<400>241

tagcaggatc cctctaagcg ytggatgcgn ttycatga 38

<210>242

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>242

tagcaggatc cctctaagcg ctggatgcgc ttccatga 38

<210>243

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>243

tagcaggatc cctctaagca atgttcagat ggatgag 37

<210>244

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>244

tagcaggatc cctctaagcc atgttcaggt ggatgag 37

<210>245

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>245

tagcaggatc cctctaagca atgtttaggt ggatgag 37

<210>246

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>246

tagcaggatc cctctaagcy atgtacaagt ggatgcg 37

<210>247

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>247

tagcaggatc cctctaagtt gtatgccgtg tctggagc 38

<210>248

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>248

tagcaggatc cctctaagtt rtatgctgtg tctggagc 38

<210>249

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>249

tagcaggatc cctctaaggt yatgttttgc atacatgg 38

<210>250

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>250

tagcaggatc cctctaaggt gatgctttgt attcatgg 38

<210>251

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>251

tagcaggatc cctctaagtt ctcsagcaac ttcatgt 37

<210>252

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>252

tagcaggatc cctctaagtt ttccagcaac ttyatgt 37

<210>253

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>253

tagcaggatc cctctaagca agcacttctg tttccccgg 39

<210>254

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>254

tagcaggatc cctctaaggc agtctttcgt acttttgct 39

<210>255

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>255

tagcaggatc cctctaaggc tagtgtgacc acrctacct 39

<210>256

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>256

tagcaggatc cctctaaggc tagtgtgacc acgctccct 39

<210>257

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>257

tagcaggatc cctctaagac caactttggc cagctctcyt 40

<210>258

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>258

tagcaggatc cctctaagac caactttggc cagctttcct 40

<210>259

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>259

tagcaggatc cctctaagac caactttgga gtcctctcgt 40

<210>260

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>260

tagcaggatc cctctaagac caacttcggt cagctctcct 40

<210>261

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>261

tagcaggatc cctctaagra gaagtgtgat ggctagcaag t 41

<210>262

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>262

tagcaggatc cctctaagaa ggagtgtgat ggctagcaag c 41

<210>263

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>263

tagcaggatc cctctaaggg stataaaatg gcttcgctca t 41

<210>264

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>264

tagcaggatc cctctaaggg ctataaaatg ckttcgctca t 41

<210>265

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>265

tagcaggatc cctctaaggg ctataaaatg gcttctctta t 41

<210>266

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>266

tagcaggatc cctctaaggt agagatcgat ggcgtctt 38

<210>267

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>267

tagcaggatc cctctaagtc gtaatatggc actgaagg 38

<210>268

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>268

aattctaata cgactcacta tagggagaga cgccatcatc atttac 46

<210>269

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>269

aattctaata cgactcacta tagggagtcg acgccatctt cattcaca 48

<210>270

<211>50

<212>DNA

<213>Artificial

<220>

<223>primer

<400>270

aattctaata cgactcacta tagggagtca tcagaatgat tgtaatttgc 50

<210>271

<211>50

<212>DNA

<213>Artificial

<220>

<223>primer

<400>271

aattctaata cgactcacta tagggagtca tcagagtgat tgtaatttgc 50

<210>272

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>272

aattctaata cgactcacta tagggagtat tacmaggtgt caaattac 48

<210>273

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>273

aattctaata cgactcacta tagggagtgt tgccaggtgt caaattac 48

<210>274

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>274

aattctaata cgactcacta tagggagtgt tcccgggtgt cagattcc 48

<210>275

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>275

aattctaata cgactcacta tagggagcct cgatgacgcc gcggt 45

<210>276

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>276

aattctaata cgactcacta tagggagcct cgatgatgcc gcggt 45

<210>277

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>277

aattctaata cgactcacta tagggagtac tgggcttctc gaagta 46

<210>278

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>278

aattctaata cgactcacta tagggagtac tgggtttctc gaagta 46

<210>279

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>279

aattctaata cgactcacta tagggagagc agtgtacggg ttaatgtt 48

<210>280

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>280

aattctaata cgactcacta tagggagtgc agtgtagggg ttaatgcgt 49

<210>281

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>281

aattctaata cgactcacta tagggagtgc ggtgtaggga ttgatgcgt 49

<210>282

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>282

aattctaata cgactcacta tagggagcca ggctttgaag ttgccgaa 48

<210>283

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>283

aattctaata cgactcacta tagggagaaa ttaatacgat tggatcaga 49

<210>284

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>284

aattctaata cgactcacta tagggagaaa ttgaaaatat tggatacgc 49

<210>285

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>285

aattctaata cgactcacta tagggagaat agagcagatt ggatcaga 48

<210>286

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>286

aattctaata cgactcacta tagggagtct gayccaattt gctctattt 49

<210>287

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>287

aattctaata cgactcacta tagggagtcc gatccaatct gttctattt 49

<210>288

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>288

aattctaata cgactcacta tagggagtct gatccaattg ttttaattt 49

<210>289

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>289

aattctaata cgactcacta tagggagtct gatccaatct gctctattt 49

<210>290

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>290

aattctaata cgactcacta tagggagtcc gatccaatct gttctattt 49

<210>291

<211>49

<212>DNA

<213>Artificial

<220>

<223>primer

<400>291

aattctaata cgactcacta tagggaggtt gatccaatct cttgtatct 49

<210>292

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>292

aattctaata cgactcacta tagggagttc ctgcatccac atagtct 47

<210>293

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>293

aattctaata cgactcacta tagggagaga ctatgtggat gcaggaa 47

<210>294

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>294

aattctaata cgactcacta tagggagtgg tatttttgac cttagc 46

<210>295

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>295

aaaaaaaaaa aaaaaaaaaa tgcgcgattg gatatgccca ggtg 44

<210>296

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>296

aaaaaaaaaa aaaaaaaaaa ttcgcgattg gatgaaccca ggtg 44

<210>297

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>297

aaaaaaaaaa aaaaaaaaaa taataccaaa tacgacccat gagg 44

<210>298

<211>42

<212>DNA

<213>Artificial

<220>

<223>probe

<400>298

aaaaaaaaaa aaaaaaaaaa tggaggaagg agaagtcgta ac 42

<210>299

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>299

aaaaaaaaaa aaaaaaaaaa acctgatcca tcgcaagcgt cacagt 46

<210>300

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>300

aaaaaaaaaa aaaaaaaaaa accgctaccg tcacaagagt cacagt 46

<210>301

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>301

aaaaaaaaaa aaaaaaaaaa gagcggcaat gccgatgaag ggctg 45

<210>302

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>302

aaaaaaaaaa aaaaaaaaaa gattcagttt attgtcatca agcatg 46

<210>303

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>303

aaaaaaaaaa aaaaaaaaaa gattatcaaa ggacgatatt gaacg 45

<210>304

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>304

aaaaaaaaaa aaaaaaaaaa tactatccar gctgcaaatg atgca 45

<210>305

<211>50

<212>DNA

<213>Artificial

<220>

<223>probe

<400>305

aaaaaaaaaa aaaaaaaaaa atattctcag atatatggat ggtatcgtgt 50

<210>306

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>306

aaaaaaaaaa aaaaaaaaaa aaagacgtat gtagattcgc tgaat 45

<210>307

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>307

aaaaaaaaaa aaaaaaaaaa gttatactga attgccatca tcagg 45

<210>308

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>308

aaaaaaaaaa aaaaaaaaaa agattaaggc tgataaaaca acagc 45

<210>309

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>309

aaaaaaaaaa aaaaaaaaaa sctcaggatg ctaaaccagt agag 44

<210>310

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>310

aaaaaaaaaa aaaaaaaaaa atccagttat gcatcgtgca tatggt 46

<210>311

<211>48

<212>DNA

<213>Artificial

<220>

<223>probe

<400>311

aaaaaaaaaa aaaaaaaaaa acattttgta tggataggtg gagaagtc 48

<210>312

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>312

aaaaaaaaaa aaaaaaaaaa atgcattttt gataaacaca ttgaaa 46

<210>313

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>313

aaaaaaaaaa aaaaaaaaaa tcaataagag caatgatttg atcaac 46

<210>314

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>314

aaaaaaaaaa aaaaaaaaaa gttcatttgg agaatttgaa gata 44

<210>315

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>315

aaaaaaaaaa aaaaaaaaaa atgctgttcg atgatatttt gcac 44

<210>316

<211>43

<212>DNA

<213>Artificial

<220>

<223>probe

<400>316

aaaaaaaaaa aaaaaaaaaa gagaacttgg tattggtctt aat 43

<210>317

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>317

aaaaaaaaaa aaaaaaaaaa gcacttggga gggggatcgc gatct 45

<210>318

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>318

aaaaaaaaaa aaaaaaaaaa tctacatttg gattggtttc tattc 45

<210>319

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>319

aaaaaaaaaa aaaaaaaaaa tctacattcg gattggtttc tattc 45

<210>320

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>320

aaaaaaaaaa aaaaaaaaaa tccacatttg gattggtttc tattc 45

<210>321

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>321

aaaaaaaaaa aaaaaaaaaa gattttgcac ttgagcttga gtttc 45

<210>322

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>322

aaaaaaaaaa aaaaaaaaaa gatttcgcac ttgagcttga gttcc 45

<210>323

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>323

aaaaaaaaaa aaaaaaaaaa gactttgcat tagagcttga attta 45

<210>324

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>324

aaaaaaaaaa aaaaaaaaaa gaagtcttcg acgtggtcag agtgca 46

<210>325

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>325

aaaaaaaaaa aaaaaaaaaa gaagttttcg acgtggtcag agtgca 46

<210>326

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>326

tagcaggatc cctctaagac cgaataataa catcaatatc g 41

<210>327

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>327

tagcaggatc cctctaagca acgttctatc tttggaacc 39

<210>328

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>328

tagcaggatc cctctaagtg cggtaggtgg ttcaac 36

<210>329

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>329

tagcaggatc cctctaaggc tgcggtcatc cgtaaaag 38

<210>330

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>330

tagcaggatc cctctaaggc ctttacccca ccaacta 37

<210>331

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>331

tagcaggatc cctctaagcc ttcggttaaa gttctaattg g 41

<210>332

<211>40

<212>DNA

<213>Artificial

<220>

<223>primer

<400>332

tagcaggatc cctctaagga taactattgg aaacgatagc 40

<210>333

<211>37

<212>DNA

<213>Artificial

<220>

<223>primer

<400>333

tagcaggatc cctctaaggg caaggaagcg aattttc 37

<210>334

<211>44

<212>DNA

<213>Artificial

<220>

<223>primer

<400>334

tagcaggatc cctctaagaa tatcgcatga gaagatgtag aaag 44

<210>335

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>335

tagcaggatc cctctaagtg gttccgttgt gaaagg 36

<210>336

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>336

tagcaggatc cctctaagac cgtcagcacc ttcatc 36

<210>337

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>337

tagcaggatc cctctaagga aggggcttgc tccttg 36

<210>338

<211>36

<212>DNA

<213>Artificial

<220>

<223>primer

<400>338

tagcaggatc cctctaagga gtgaagcggc aaaagc 36

<210>339

<211>39

<212>DNA

<213>Artificial

<220>

<223>primer

<400>339

tagcaggatc cctctaagtt ggatttgcat tgcgaactg 39

<210>340

<211>41

<212>DNA

<213>Artificial

<220>

<223>primer

<400>340

tagcaggatc cctctaagtt tgttactgtg gtagatacta c 41

<210>341

<211>38

<212>DNA

<213>Artificial

<220>

<223>primer

<400>341

tagcaggatc cctctaaggt cgatgtatgt cttgttgc 38

<210>342

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>342

aattctaata cgactcacta tagggagctc accaactatc tgatacg 47

<210>343

<211>51

<212>DNA

<213>Artificial

<220>

<223>primer

<400>343

aattctaata cgactcacta tagggagagt tttgcactta actttaattt g 51

<210>344

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>344

aattctaata cgactcacta tagggagttg tcgcggattt gcaac 45

<210>345

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>345

aattctaata cgactcacta tagggagagc aggttcaagc gaaccaa 47

<210>346

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>346

aattctaata cgactcacta tagggagtac cgaatgtggc gatatttg 48

<210>347

<211>46

<212>DNA

<213>Artificial

<220>

<223>primer

<400>347

aattctaata cgactcacta tagggagagc ccagagcatc taaagg 46

<210>348

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>348

aattctaata cgactcacta tagggagatc tcacagtgaa gcaattg 47

<210>349

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>349

aattctaata cgactcacta tagggagccc tatttctaga ggctatcc 48

<210>350

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>350

aattctaata cgactcacta tagggagtga tacgtcgcac cctcatc 47

<210>351

<211>48

<212>DNA

<213>Artificial

<220>

<223>primer

<400>351

aattctaata cgactcacta tagggagggc taaacatcat agtcttgg 48

<210>352

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>352

aattctaata cgactcacta tagggagctc cgtgtagtcc agcag 45

<210>353

<211>50

<212>DNA

<213>Artificial

<220>

<223>primer

<400>353

aattctaata cgactcacta tagggaggta ttagctacag tttcccgtag 50

<210>354

<211>47

<212>DNA

<213>Artificial

<220>

<223>primer

<400>354

aattctaata cgactcacta tagggagggc ccaaagatac cttcttc 47

<210>355

<211>47

<212>DNA

<213>Artificial

<220>

<223>probe

<400>355

aattctaata cgactcacta tagggagcca aggtcggcac ttctaac 47

<210>356

<211>52

<212>DNA

<213>Artificial

<220>

<223>probe

<400>356

aattctaata cgactcacta tagggagaaa taaactgtaa atcatattcc tc 52

<210>357

<211>52

<212>DNA

<213>Artificial

<220>

<223>probe

<400>357

aattctaata cgactcacta tagggagtat attcatgcaa tgtaggtgta tc 52

<210>358

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>358

aaaaaaaaaa aaaaaaaaaa cgcgtttgcg acgcttttgg atggg 45

<210>359

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>359

aaaaaaaaaa aaaaaaaaaa agtcttgtgt tcaacttgca atgga 45

<210>360

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>360

aaaaaaaaaa aaaaaaaaaa tagtacgaac tgttgccttg gaaga 45

<210>361

<211>44

<212>DNA

<213>Artificial

<220>

<223>probe

<400>361

aaaaaaaaaa aaaaaaaaaa ctgtattgct cgtccgtagg cttc 44

<210>362

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>362

aaaaaaaaaa aaaaaaaaaa cggttaaggg agagtctatg tgatat 46

<210>363

<211>45

<212>DNA

<213>Artificial

<220>

<223>primer

<400>363

aaaaaaaaaa aaaaaaaaaa agagggtagc aataacaggt ccgtg 45

<210>364

<211>48

<212>DNA

<213>Artificial

<220>

<223>probe

<400>364

aaaaaaaaaa aaaaaaaaaa aataccgcat aagagtaatt aacacatg 48

<210>365

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>365

aaaaaaaaaa aaaaaaaaaa aacgcgtggg taatctgcct ttgaga 46

<210>366

<211>46

<212>DNA

<213>Artificial

<220>

<223>probe

<400>366

aaaaaaaaaa aaaaaaaaaa agtcgctctt tgtggcgacg cttttg 46

<210>367

<211>45

<212>DNA

<213>Artificial

<220>

<223>probe

<400>367

aaaaaaaaaa aaaaaaaaaa cgcatggttc cgttgtgaaa ggcgc 45

Claims (18)

1. A method for detecting bacterial species of bacteria, wherein the bacterial species selected from Staphylococcus (Staphylococcus) bacteria, Streptococcus (Streptococcus) bacteria, Klebsiella (Klebsiella) bacteria, Escherichia (Escherichia) ) bacteria, Mycobacterium (Mycobacterium) bacteria, Legionella (Legionella) bacteria, Vibrio (Vibrio) bacteria, Bacillus (Bacillus) bacteria, Neisseria (Neisseria) bacteria, Campylobacter ( Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Listeria, Salmonella and Yersinia ( More than 2 kinds of bacterial strains in Yersinia bacteria are detection objects, comprising the following steps: (a) contacting a test sample that may contain a nucleic acid of a bacterium to be detected with two or more nucleic acid molecules that hybridize to at least a part of the DnaJ genes of two or more bacterial species to be detected, and (b) detecting whether said nucleic acid molecule hybridizes to nucleic acid in said test sample. 2. detection method according to claim 1, wherein the strain of bacteria is selected from Streptococcus pyogenes (Streptococcus pyogenes), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus pneumoniae (Streptococcus pneumoniae), Staphylococcus aureus ( Staphylococcus aureus), Klebsiella pneumoniae, Escherichia coli, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Mycoplasma genitarium, Vibrio cholerae, para Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio fluvialis, Vibrio alginolyticus, Neisseria meningitides, Neisseria gonorrhoeae gonorrhoeae), Bacillus cereus, Chlamydia trachomatis, Chlamydophila pneumoniae and Campylobacter jejuni. 3. The method according to claim 1 or 2, wherein the detection object is selected from Klebsiella (Klebsiella) bacteria, Escherichia (Escherichia) bacteria, Vibrio (Vibrio) bacteria, spores Bacillus, Neisseria, Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Listeria Two or more bacterial species of Listeria, Salmonella, and Yersinia. 4. The detection method according to any one of claims 1 to 3, wherein the nucleic acid molecule comprises one or more than two kinds of DNA extracted by comparing the base sequences of the DnaJ genes of bacterial strains belonging to the same bacterial species. A bacterial species-specific nucleotide sequence or a nucleotide sequence substantially identical to the nucleotide sequence. 5. The detection method according to any one of claims 1 to 4, wherein the (a) step uses at least one first marker sequence and a base sequence that selectively anneals to a part of the DnaJ gene. performing a polymerase chain reaction with a primer set, and at least one second primer set having a marker sequence substantially identical to that of the first primer set, and using the first primer set to amplify the DnaJ gene At least a part of the amplification product amplified by the first primer set is amplified using the second primer set. 6. The amplification method according to claim 5, wherein said polymerase chain reaction step is performed in a single PCR reaction cycle. 7. The detection method according to any one of claims 1 to 6, wherein, When the bacterium comprises Streptococcus pyogenes, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 1 to 4 and SEQ ID NO: 89 to 90 or substantially the same base sequence; When the bacterium comprises Streptococcus agalactiae, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 5-8 and SEQ ID NO: 91-92 or a base sequence substantially identical thereto; When the bacterium comprises Streptococcus pneumoniae, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NOs: 9-12 and SEQ ID NOs: 93-94 or substantially the same base sequence; When the bacterium comprises Staphylococcus aureus, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NOs: 13-16 and SEQ ID NOs: 97-98 or a base sequence thereof on the same base sequence; When the bacterium comprises Klebsiella pneumoniae (Klebsiella pneumoniae), the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NOs: 17-19 and SEQ ID NOs: 109 and 110 or a base sequence substantially identical thereto; When the bacterium comprises Escherichia coli (Escherichia coli), the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 21 to 24 and SEQ ID NO: 101 and 102 or is substantially the same base sequence; When the bacterium comprises Mycobacterium tuberculosis (Mycobacterium tuberculosis), the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NOs: 25-28 and SEQ ID NOs: 103-104 or a base sequence thereof on the same base sequence; When the bacterium comprises Mycobacterium spp. other than M. tuberculosis (M.tuberculosis) group, the nucleic acid molecule comprises a nucleic acid molecule selected from among SEQ ID NO: 29-30 and SEQ ID NO: 131-133 1 or more base sequences or base sequences substantially identical thereto; When the bacterium comprises Legionella pneumophila, the nucleic acid molecule comprises one or more nucleotide sequences selected from SEQ ID NO: 35-38 and SEQ ID NO: 115-116 or a base sequence thereof on the same base sequence; When the bacterium comprises Vibrio cholerae, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 43-46 and SEQ ID NO: 127-128 or substantially the same base sequence; When the bacterium comprises Vibrio mimicus, the nucleic acid molecule comprises one or more bases selected from SEQ ID NO: 43, SEQ ID NO: 47-49 and SEQ ID NO: 125-126 base sequence or a base sequence substantially identical thereto; When the bacterium comprises Vibrio parahaemolyticus (Vibrio parahaemolyticus), the nucleic acid molecule comprises one or more selected from SEQ ID NO: 43, SEQ ID NO: 50-52 and SEQ ID NO: 123-124 a base sequence or a base sequence substantially identical thereto; When the bacterium comprises Vibrio vulnificus, the nucleic acid molecule comprises one or more base sequences of SEQ ID NO: 43, SEQ ID NO: 53-55 and SEQ ID NO: 119-120 or a base sequence substantially identical thereto; When the bacterium comprises Vibrio fluvialis, the nucleic acid molecule comprises 1 selected from SEQ ID NO: 43, SEQ ID NO: 134, SEQ ID NO: 56-57 and SEQ ID NO: 117-118. one or more base sequences or base sequences substantially identical thereto; When the bacterium comprises Vibrio alginolyticus, the nucleic acid molecule comprises one or more selected from SEQ ID NO: 43, SEQ ID NO: 58-60 and SEQ ID NO: 121-122 a base sequence or a base sequence substantially identical thereto; When the bacterium comprises Bacillus cereus, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NOs: 61-66 and SEQ ID NOs: 87-88 or a base sequence thereof on the same base sequence; When the bacterium comprises Campylobacter jejuni, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 67-70 and SEQ ID NO: 129-130 or substantially the same base sequence; When the bacterium comprises Neisseria meningitidis, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 83-86 and SEQ ID NO: 111-112 or a base sequence substantially identical thereto; When the bacterium comprises Neisseria gonorrhoeae (Neisseria gonorrhoeae), the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 79-82 and SEQ ID NO: 113-114 or a combination thereof substantially the same base sequence; When the bacterium comprises Chlamydia trachomatis, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 75-78 and SEQ ID NO: 109-110 or is substantially the same base sequence; When the bacterium comprises Chlamydophila pneumoniae, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 39-42 and SEQ ID NO: 107-108 or substantially the same base sequence; When the bacterium comprises Mycoplasma genitarium, the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 71-74 and SEQ ID NO: 105-106 or a base sequence substantially identical thereto ; When the bacterium comprises Mycoplasma pneumoniae (Mycoplasma pneumoniae), the nucleic acid molecule comprises one or more base sequences selected from SEQ ID NO: 31-34 and SEQ ID NO: 95-96 or is substantially the same base sequence. 8. A nucleic acid molecule kit for detection and identification of bacteria, wherein, selected from Streptococcus (Streptococcus) bacteria, Staphylococcus (Staphylococcus) bacteria, Enterococcus (Enterococcus) bacteria, Peptostreptococcus (PeptoStreptococcus) bacteria ) bacteria, Klebsiella bacteria, Escherichia bacteria, Enterobacter bacteria, Legionella bacteria, Neisseria bacteria, Bacillus, Helicobacter, Corynebacterium, Pseudomonas, Burkholderia, Haemophilus ( Haemophilus, Bacteroides, Enterococcus, Nocardia, Prevotella, Neisseria, Morakot Moraxella, Flavobacterium, Clostridium, Treponema, Sphingobacterium, Leptospira , Campylobacter, Listeria, Salmonella and Yersinia are the objects of detection and identification, Wherein the kit comprises a nucleic acid molecule hybridized to at least a part of the DnaJ gene of the bacterium to be identified. 9. The kit according to claim 8, wherein the nucleic acid molecule comprises at least one first primer set having a marker sequence and a base sequence that selectively hybridizes to a part of the DnaJ gene, and at least one A second primer set having a marker sequence substantially identical to that of the first primer set. 10. A solid phase in which two or more nucleic acid molecules selected from the nucleic acid molecules constituting the nucleic acid molecule kit according to claim 8 or 9 are immobilized. 11. A method for amplifying at least one target nucleic acid, comprising the steps of: using at least one first primer set having a marker sequence and a base sequence that selectively anneals to a part of the target nucleic acid, and at least performing a polymerase chain reaction with a second primer set having a marker sequence substantially identical to the marker sequence of the first primer set, The polymerase chain reaction step performs amplifying the target nucleic acid using the first primer set and amplifying an amplification product obtained by amplifying using the first primer set using the second primer set, The first primer set uses a part of the DnaJ gene of the microorganism as the target nucleic acid. 12. The amplification method according to claim 11, wherein said polymerase chain reaction step is carried out in a single PCR reaction cycle. 13. The amplification method according to claim 11 or 12, wherein the primer concentration of the second primer set is 10 times to 50 times that of the first primer set. 14. The amplification method according to claim 11, wherein the primer concentration of the first primer set is 0.005 μM to 0.1 μM, and the primer concentration of the second primer set is the primer concentration of the first primer set. The concentration is more than 10 times and less than 50 times, The amplified product obtained by using the second primer set to amplify is a nucleic acid of 250 bases or less, The polymerase chain reaction step implements a single PCR reaction cycle. 15. The amplification method according to any one of claims 11 to 14, wherein the first primer set will be selected from Staphylococcus (Staphylococcus) bacteria, Streptococcus (Streptococcus) bacteria, Serratia ( Serratia, Klebsiella, Escherichia, Mycobacterium, Legionella, Vibrio, Bacillus, Neisseria, Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Lys A part of the DnaJ gene of two or more bacterial species among Listeria, Salmonella, and Yersinia was used as the target nucleic acid. 16. A method utilizing multiplex PCR to diagnose pathogenic microorganisms, said pathogenic microorganisms being selected from Staphylococcus (Staphylococcus) bacteria, Streptococcus (Streptococcus) bacteria, Serratia (Serratia) bacteria, Klebsiella Klebsiella, Escherichia, Mycobacterium, Legionella, Vibrio, Bacillus, Neisseria, Campylobacter, Chlamydia, Chlamydophila, Mycoplasma, Listeria, Two or more of Salmonella and Yersinia bacteria, The method comprises using at least one first primer set and at least one second primer set to perform polymerase chain reaction on a test sample possibly containing nucleic acid from said pathogenic microorganism, and detecting an amplification product containing said target nucleic acid, The first primer set has a marker sequence and a nucleotide sequence that selectively anneals to the target nucleic acid of the DnaJ gene possessed by the pathogenic microorganism, and the second primer set has a marker sequence substantially identical to the marker sequence of the first primer set. the same sequence of markers. 17. The method of claim 16, wherein the polymerase chain reaction step is performed in a single PCR reaction cycle. 18. A kit comprising a primer set for amplifying at least one target nucleic acid of a pathogenic microorganism by multiplex PCR, the kit comprising: The first primer set and the second primer set, The first primer set has a marker sequence and a nucleotide sequence that selectively anneals to the target nucleic acid of the DnaJ gene possessed by the pathogenic microorganism, and the second primer set has a marker sequence substantially identical to the marker sequence of the first primer set. the same sequence of markers.

Images