CN105154438B - To Hafnia alvei G5907, G5908, G5913, nucleotide special G5916 and its application - Google Patents

Abstract

The present invention relates to nucleotides specific to Hafnia alvei G5907, G5908, G5913, G5916 and applications thereof, wherein the nucleotides include one of the nucleotides shown in SEQ ID NO: 1‑8. These nucleotides can be used to prepare PCR kits and gene chips for detecting Hafnia alvei. The present invention is Hafnia alvei G5907, G5908, G5913, G5916 specific nucleotides and a PCR kit and a gene chip containing the nucleotides. The technical operation method is simple, the detection period is short, and the detection cost is low. The method has high accuracy and high sensitivity and is easy for industrialized production.

Description

Nucleotides specific to Hafnia alvei G5907, G5908, G5913, G5916 and its application

technical field

The present invention relates to nucleotides specific to Hafnia alvei G5907, G5908, G5913, G5916, in particular to nucleotides specific to a single gene in the O antigen gene cluster of Hafnia alvei G5907, G5908, G5913, G5916 Glycolic acid and its application.

Background technique

Hafnia alvei is a kind of Enterobacteriaceae. It is straight rod-shaped, non-capsulated, motile, facultative anaerobic, and has two types of metabolism: respiration and fermentation. It is more common in food such as respiratory tract, intestinal tract, animal intestinal tract and meat and dairy products. Hafnia alveoli is an opportunistic pathogen that can cause bacteremia, respiratory tract infection, urinary tract infection and other infections in people with low immunity. It may be related to gastroenteritis. In addition, It can also cause catarrhal enteritis and hepatosplenomegaly in laying hens, animal diseases such as zoonotic septicemia in Bulgarian rainbow trout.

Bacterial typing and identification methods mainly include traditional phenotypic methods, serological methods and molecular identification methods. However, with the development of molecular biology, there are certain problems in the traditional serotyping and identification methods. For example, the diagnostic method of serotyping requires a large amount of antiserum, and the antiserum is generally incomplete and insufficient. Antisera also present some difficulties in preparation and storage. On the other hand, the serotyping method takes a long time, has low sensitivity, high missed detection rate, and poor accuracy. There are often cross-reactions between antisera produced by different O antigens. Therefore, establishing a serum identification method based on molecular biology technology has become a development direction.

The surface antigens of Hafnia alvei include both O antigen and H antigen, but the O antigen is mainly used for typing, and the O antigen research is relatively thorough. The official serotype scheme for Hafnia hives that has been determined so far was proposed by a laboratory at the Mechnikov Institute of Vaccine and Serum Research in Moscow, Russia, and 39 O and 36 H types have been confirmed. This taxonomy has been the method of choice for studying epidemiological relationships between strains and is particularly useful in comparing strains from different places at different times. More and more people pay more and more attention to its molecular identification. Molecular biology detection technology has the advantages of fast speed and easy large-scale use, and it is currently the internationally recognized development direction of pathogenic bacteria detection.

In recent years, more and more molecular techniques have been used for typing, identification, detection and disease diagnosis of pathogenic bacteria, including sequence analysis of transcribed spacer (ITS), random amplified length polymorphism (RAPD) analysis, ribosomal DNA restriction Sexual fragment length polymorphism (RFLP) analysis, etc. Molecular biology methods can not only be used for rapid serotyping screening of Hafnia alvei, stable identification results can make up for the lack of phenotypic characterization methods. Compared with traditional detection technologies, these polymerase chain reaction (PCR)-based molecular detection technologies do not require the isolation and pure cultivation of pathogenic bacteria, and have the advantages of rapidity, sensitivity, and strong specificity.

Polymerase chain reaction technology (Polymerase chain reaction, referred to as PCR technology) is currently being recognized and promoted as a microbial detection technology. Compared with traditional methods, this technology has the advantages of high throughput, fast detection speed, strong specificity, and high sensitivity. Simply perform a simple enrichment process on the sample, and then prepare the bacterial DNA template by centrifugation and lysis, and then the target sequence can be amplified in the PCR process mediated by highly specific primers to detect whether the sample contains the pathogen to be detected. The purpose of pathogenic microorganisms. The PCR amplification process only takes 1.5 hours. This undoubtedly greatly improves the work speed for the inspection and quarantine department and clinical inspection, and also reduces the work cost.

Contents of the invention

The object of the present invention is to provide that the present invention relates to Hafnia alvei G5907, G5908, G5913, G5916 specific nucleotide, it is characterized in that described nucleotide has:

1) One of the nucleotides shown in SEQ ID NO:1-8;

2) One of the nucleotides complementary to the nucleotides shown in SEQ ID NO:1-8; said SEQ ID NO:1-8 is as follows:

The present invention also provides a PCR kit, including PCR primers, dNTP, buffer and DNA polymerase, and the PCR primer is one of the nucleotides shown in SEQ ID NO: 1-8. The kit also includes the following reagents: 30 μl of 10mM dNTP; 50 μl of 10× enzyme-specific reaction buffer; 5 μl of 5 U/μl heat-resistant DNA polymerase; 10 μl of mixed primers (upstream and downstream primers specific to each standard strain, singular number is Upstream primers, double numbers are downstream primers, G5908 is SEQ ID NO:1-2; G5908 is SEQ ID NO:3-4; G5913 is SEQ ID NO:5-6; G5916 is SEQ ID NO:7-8); Positive control substance 10μl; negative control substance 10μl; ddH ₂ O 5ml.

Wherein the PCR primer is preferably one of the nucleotides shown in SEQ ID NO: 1-8.

The present invention further discloses the preparation of PCR kits for different strains G5907, G5908, G5913 and G5916 of Hafnia alvei G5907, G5908, G5913 and G5916 specific SEQ ID NO: 1-8 nucleotides.

The Hafnia alvei mentioned in the present invention refers to the crude extract sampled from the culture of clinical specimens such as sepsis, urinary tract infection, wound infection or the crude extract of the pure culture of Hafnia alvei. Collecting Hafnia alvei and extracting the genome is prepared by conventional methods.

For the PCR kit for Hafnia alvei, the entire detection step includes sample pretreatment-amplification-electrophoresis detection results. The primers and reagents required for the PCR reaction system have been added to the amplification tube in advance. The user only needs to add the pretreated sample to the amplification tube to start the amplification reaction, and complete the detection work simply and quickly.

The present invention also provides a liquid-phase detection chip, including liquid-phase magnetic beads and oligonucleotide probes connected to the liquid-phase magnetic beads, and corresponding primers corresponding to the fragments where the corresponding probes are located; wherein the nucleotide Preferred are the nucleotides shown in SEQ ID NO: 1-8.

The present invention also provides a microarray, which includes the above-mentioned nucleotides; wherein the nucleotides are preferably nucleotides as shown in SEQ ID NO: 1-8.

The present invention further discloses the use of nucleotides specific to Hafnia alvei G5907, G5908, G5913, and G5916 in preparing PCR kits for detecting Hafnia alvei and gene chips for detecting Hafnia alvei , Detection of Hafnia hives microarray application. The detection of Hafnia alvei refers to the detection of bacteria that cause bronchitis, pneumonia, urinary tract infection, wound infection and sepsis.

Compared with the prior art, compared with the prior art, the present invention has the following advantages:

(1) Strong practicability

A PCR reaction system established by the invention can detect Hafnia hives, provide specific primers used for serotype detection, and use the PCR method to detect clinical specimens.

(2) High accuracy

In the present invention, through the PCR reaction of each serotype-specific gene of Hafnia alvei, one band is obtained for each sample, and the obtained target fragment is compared with the known length to obtain Hafnia alvei Corresponding strain number.

(3) The detection cost is relatively low

It can be popularized and applied in food hygiene supervision, environmental monitoring, commodity monitoring, inspection and quarantine and other fields, and provide a technical model for other different pathogenic bacteria detection combinations.

Description of drawings:

Fig. 1 shows the G5907-specific primers of the present invention to detect the electrophoresis results of Hafnia alvei and other standard strains. The wzy gene P1 and P2 target bands are 231bp, and the rest of the strains do not have any bands. The specific strain information is shown in Table 2;

Fig. 2 shows the identification electrophoresis results of G5907-specific primer species specificity of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected, without any bands, and the specific strain information is shown in Table 2;

Fig. 3 shows the electrophoresis results of the wzy gene P3 and P4 primers of G5908 of the present invention to detect Hafnia alvei and other standard strains, the target band is 269bp, and the specific strain information is shown in Table 2;

Fig. 4 shows the identification electrophoresis results of species specificity of the wzy gene P3 and P4 primers of G5908 of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected, without any bands. For specific strain information, see surface;

Fig. 5 shows the electrophoresis results of the wzy gene P5 and P6 primers of G5913 of the present invention to detect Hafnia alvei and other standard strains. The target band is 189bp, and the rest of the strains do not have any bands. The specific strain information is shown in Table 2;

Fig. 6 shows the identification electrophoresis results of species specificity of the wzy gene P5 and P6 primers of G5913 of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected, without any bands. For specific strain information, see Table 2;

Fig. 7 shows the electrophoresis results of the wzy gene P5 and P6 primers of G5916 of the present invention to detect Hafnia alvei and other standard strains. The target band is 235bp, and the rest of the strains do not have any bands. The specific strain information is shown in Table 2;

Fig. 8 shows the electrophoresis results of species-specific identification of wzy gene P5 and P6 primers of G5916 of the present invention, in which 6 strains of Vibrio, 1 strain of Salmonella, and 1 strain of Escherichia coli were detected without any bands. For specific strain information, see Table 2;

Figure 9 shows the electrophoresis results of the corresponding serotypes amplified with Hafnia alvei G5907, G5908, G5913, and G5916 specific primers respectively, and the specific strain information is shown in Table 2;

Among them: Hafnia alvei G5907, G5908, G5913, and G5916 represent the amplification results of the corresponding serotype primers, and the first lane is 2000bp DNA marker or 1kb plus DL.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, the conventional conditions are generally followed, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). Among them, Hafnia hives came from Warsaw, Poland (PCM, Polish Collection of Microorganisms at IITD PAN, Wroclaw).

Embodiment 1 : the extraction of genome

Hafnia alveoli was cultured in nutrient broth medium at 37°C, the bacteria were collected, and the genome was extracted. The specific steps are as follows:

The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg/ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg/ml proteinase K, 15ul10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg/ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol for extraction, take the supernatant, and then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether to remove residual phenol. Precipitate DNA with 2 times the volume of ethanol in the supernatant, roll out the DNA with glass wool and wash the DNA with 70% ethanol, and finally resuspend the DNA in 30ul TE. Genomic DNA was detected by 0.4% agarose gel electrophoresis.

Embodiment 2: sequence deciphering

The genomes of Hafnia alvei G5907, G5908, G5913, and G5916 standard strains were extracted, and the whole genome sequence of each standard genome of Hafnia alvei was obtained by Solexapair-end sequencing technology to obtain the sequence of the O serotype. Blast and PSI-Blast for sequence comparison, TMHMM Server2.0 program for transmembrane structure prediction, ClustalW program for sequence alignment and screening of conserved and specific gene fragments, and finally the O antigen gene clusters of each serotype of Hafnia alvei Sequence and Deciphering Results.

Embodiment 3 : primer design

According to the deciphering of the gene cluster and the results of database comparison, it was found that the wzy and wzx genes are specific genes for the O antigen of Hafnia alvei, so the specific segment of the gene was selected to design specific primers. Since wzy is more specific, the wzy gene is mainly used as the target gene.

Primer design is a core part of this invention. Primers were designed according to the specific genes described in the literature. The two genes wzx and wzy are relatively specific genes in the O antigen gene cluster of Hafnia alvei, which can be used as target genes for serotype identification. The above genes were introduced into Primer Premier 5 for primer design. The length of the primers should preferably be between 18 and 24 bp, and the Tm value should be between 53 and 58°C. A pair of primers are designed for each gene, with a single target band.

After the primers are designed, BLAST is performed in Genbank. The designed primers should not have too high sequence similarity with other related bacteria, so as to ensure that the primers can only amplify at their predetermined positions, and not with other related bacteria or bacteria. Relative bacteria in the environment where the specimen was collected did not produce a positive reaction. This point is very important to avoid the generation of non-specific bands and the success or failure of the experiment.

The designed primers are shown in Table 1.

Table 1 Primer sequences used for PCR

Embodiment 4 : the screening of specific primer

The standard strains of Hafnia alvei G5907, G5908, G5913, and G5916 were collected, including: 6 strains of Vibrio, 1 strain of Salmonella and 1 strain of Escherichia coli to verify the specificity of the primers. The strain numbers and sources are shown in the table 2. At the same time, provide the electronic version of the certificate of entry and exit of Hafnia hives. The paper version of the stamped document is in the Tianjin Municipal Health Bureau. It is used for approval. See other supporting documents:

Table 2 Strains used for specific detection

The PCR system used for gene identification primer screening is 0.4 μl of 5 μM primers, 2.5 μl of 10× enzyme-specific reaction buffer, 0.25 μl of 10 mM dNTP, 0.2 μl of 5 U/μl thermostable DNA polymerase, and 3 μl of the sample template to be tested to 0.2 ml of thin-walled PCR tubes, and finally make up to 25 μl of ddH ₂ O. All primers obtained positive results in their respective strains, and did not obtain any PCR product bands in other groups.

The reaction cycle parameters on the PCR instrument in this step include the denaturation, renaturation, temperature and time of extension, and the number of cycles of DNA, specifically:

One cycle of the pre-treatment process necessary for the denaturation to reach the required temperature in the early stage is 95 ° C for 5 minutes;

The denaturation temperature and time are 95°C, 45 seconds;

The annealing temperature and time are 53°C/58°C, 45 seconds;

The extension temperature and time are 72°C, 1 minute;

The number of cycles of denaturation, renaturation, and extension is 35 cycles;

The temperature and time for one cycle to stabilize the amplification product are 72°C for 5 minutes

Among them, G5907, G5908, G5913, and G5916 can be amplified at 53-58°C. The above steps are electrophoresis amplification products in the electrophoresis equipment. The specific steps for recording the results are:

Take 2-5 μl of the amplification product and mix it with 6× bromophenol blue loading buffer at a volume ratio of 1:1;

Load the mixture on a 1.0% agarose gel;

Perform agarose gel electrophoresis at 120v constant voltage for about 30 minutes, and use DL2000 Marker as a control;

Observe and record the results.

After the basic PCR reaction, the work of primer screening is basically completed, and the necessary length adjustment has little influence on the overall reaction conditions. The sequences of the primers used in the present invention are all summarized in Table 1.

Table 1 Primer sequences used for PCR

Embodiment 6: Preparation and application of PCR detection kit

1. The composition of the PCR kit:

dNTP (10mM) 30μl;

10×Buffer (10×Enzyme Specific Reaction Buffer) 50μl;

Taq polymerase (5U/μl thermostable DNA polymerase) 5μl;

PCR primer (5μM) SEQ ID-NO:1-8 10μl;

Positive control substance (KP) 10μl;

Negative control substance (KN) 10μl;

ddH ₂ O 5ml;

Each kit can be used to detect 10 samples.

Among them, 10×Buffer, dNTP, and Taq polymerase were provided by Bao Biological Engineering Co., Ltd.; primers were self-designed sequences provided to Shanghai Yingjun Biotechnology Company for synthesis; positive control substances, negative control substances and ddH ₂ O were prepared by ourselves.

2. Instruments and equipment

Among them, 10×Buffer, dNTP, and Taq polymerase were provided by Shanghai Sangong; primers were self-designed sequences provided to Shanghai Yingjun Biotechnology Company for synthesis; positive control substances, negative control substances and ddH2O were prepared by us. Experimental equipment PCR instrument (also known as DNA thermal cycle amplification instrument), electrophoresis equipment (including electrophoresis instrument and electrophoresis tank), gel imager, -20 ℃ refrigerator, high-speed centrifuge, micropipette and 0.2ml PCR thin film wall pipe.

3. Specific examples of the use of PCR kits

The PCR detection method that uses above-mentioned PCR kit to detect Hafnia alvei comprises the following steps:

(1) Extract the template of the environmental sample to be tested;

(2) Add, dNTP, 10×Buffer, Taq polymerase, primers, sample template to be tested and ddH ₂ O into the PCR thin-walled tube and mix well;

(3) Amplify the mixture in the thin-walled PCR tube on the PCR instrument;

(4) Electrophoresis amplifies the product in the electrophoresis equipment, and records the result;

(5) Analyze and judge the results.

The environmental sample template in the above step (1) is the crude extract of the culture of Hafnia alveoli, which is sampled from clinical specimens such as sepsis, urinary tract infection, and wound infection, and the crude extract of the pure culture of Hafnia alveoli Either pure DNA, or positive and negative controls.

The extraction method of the environmental sample template in the above step (1) is:

Take 1.5ml of the culture, centrifuge at 12000rpm for 1 minute, and remove the supernatant;

Take 500 μl of ddH ₂ O to resuspend the pellet, centrifuge at 8000 rpm for 5 minutes, remove the supernatant, and dry;

Take 100 μ lddH ₂ O to resuspend the precipitate, and bathe in boiling water at 100°C for 10 minutes;

After placing on ice for 10 minutes, centrifuge at 12000rpm for 2 minutes;

⑤ Take 3μl middle layer supernatant as PCR template

The reaction cycle parameters on the PCR instrument in the above step (3) include the temperature and time of DNA denaturation, renaturation, extension, and number of cycles, specifically:

One cycle of the pre-treatment process necessary for the denaturation to reach the required temperature in the early stage is 95 ° C for 5 minutes;

The denaturation temperature and time are 95°C, 45 seconds;

The annealing temperature and time are 53°C/58°C, 1 minute;

The extension temperature and time are 72°C, 1 minute;

The number of cycles of denaturation, renaturation, and extension is 35 cycles;

The temperature and time for one cycle to stabilize the amplification product was 72°C for 5 minutes.

The above step (4) electrophoresis amplifies the product in the electrophoresis equipment, and the specific steps for recording the results are:

Take 2-5 μl of the amplification product and mix it with 6× bromophenol blue loading buffer at a volume ratio of 5:1;

Load the mixture on a 1.0% agarose gel;

Perform agarose gel electrophoresis at 120v constant voltage for about 30 minutes, and use DL2000 Marker as a control;

Observe and record the results.

The present invention configures an industrially produced PCR kit capable of detecting Hafnia hives, and combines the components needed for the PCR detection method. Rapid, sensitive, and simple detection can be carried out by operating the sequence. The dosage and concentration of each component in the kit are all experimental results. The test equipment used to detect Hafnia alveoli with this kit is simple and the detection cost is low. .

The purpose of using positive and negative control substances is to control the entire operation process in order to make accurate judgments. If it contains the strain type of Hafniales alveoli, the band at the same position as the positive control can be observed from the electrophoresis results; with this strip.

The amount of reagents in the kit used for a detection test of the present invention is shown in the following table 3, and the amount of DNA template is 3 μl

Table 3 The amount of reagents in the kit used in a detection test

The thermostable DNA polymerase in the present invention is Taq enzyme.

The above-mentioned positive control substance is a sample that has been determined to be the standard strain of Hafnia alvei, and the negative control substance is a sample that has been confirmed by the laboratory to be not Hafnia alvei.

This PCR kit uses the bacterial suspension of Hafnia alvei for PCR amplification, which is consistent with the result obtained by using the extracted DNA as a template. There is no difference in sensitivity and specificity. In this way, the extraction step of template DNA can be omitted, and the operation method can be simplified. At the same time, compared with conventional biochemical detection methods, the sample to be tested in this method can be directly the culture solution of clinical samples, or the detection can be performed by simply separating and culturing the test sample, thus saving manpower and material resources.

4. Provision of samples to be tested

Hafnia alvei G5907, G5908, G5913, G5916 standard strains, 6 other strains of Vibrio, 1 strain of Salmonella and 1 strain of Escherichia coli were collected to verify the specificity of the primers. For the strain numbers and sources, see Table 2.

The above is only the operation and implementation method of the present invention, and does not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention still belong to within the scope of the technical solutions of the present invention.

SEQUENCE LISTING

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Claims (2)

1. a kind of primer pair, it is characterised in that the nucleotide sequence of the primer pair such as SEQ ID NO:Shown in 1-2.

2. a kind of PCR kit, including primer pair described in claim 1, dNTP, buffer solution and archaeal dna polymerase.