[go: up one dir, main page]

WO2011004725A1 - Procédé de détection de bactéries pathogènes, et kit d'utilisation de ce procédé - Google Patents

Procédé de détection de bactéries pathogènes, et kit d'utilisation de ce procédé Download PDF

Info

Publication number
WO2011004725A1
WO2011004725A1 PCT/JP2010/060940 JP2010060940W WO2011004725A1 WO 2011004725 A1 WO2011004725 A1 WO 2011004725A1 JP 2010060940 W JP2010060940 W JP 2010060940W WO 2011004725 A1 WO2011004725 A1 WO 2011004725A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
primer set
targeting
primer
streptococcus
Prior art date
Application number
PCT/JP2010/060940
Other languages
English (en)
Japanese (ja)
Inventor
克英 三宅
裕之 綱島
新太 片山
Original Assignee
国立大学法人名古屋大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人名古屋大学 filed Critical 国立大学法人名古屋大学
Publication of WO2011004725A1 publication Critical patent/WO2011004725A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a pathogen detection technology. Specifically, the present invention relates to a pathogen detection method and a kit used therefor. In particular, the present invention relates to a method for simultaneously detecting two or more types of pathogenic bacteria and a kit used therefor.
  • This application claims priority based on Japanese Patent Application No. 2009-161351 filed on Jul. 8, 2009, the entire contents of which are incorporated by reference.
  • a wide variety of pathogenic bacteria may exist in soil samples and sewage samples. In such a case, in order to ensure safety, it is desired to rapidly detect various pathogenic bacteria at once. Moreover, since it is naturally expected that microorganisms other than the pathogenic bacteria are mixed, it is necessary to increase the specificity for the pathogenic bacteria. Conventional detection methods are difficult to meet these demands. For example, in the case of detection method using 16S rRNA (Patent Document 2), since 16S rRNA gene is present in all bacteria, no matter how much the sequence is used to increase the specificity, there is a possibility that nonspecific amplification will occur. I can't deny it.
  • the detection method using a probe of a gene (toxin gene or the like) specific for each pathogen can ensure specificity, but is not appropriate as a universal pathogen detection method. Then, this invention makes it a subject to detect the pathogenic microbe which can exist in a sample simply, rapidly, and specifically.
  • the present inventors paid attention to a biosynthetic gene group of capsular polysaccharides specific to pathogenic bacteria.
  • Capsular polysaccharides are present in almost all pathogens and are closely related to pathogenicity.
  • the capsular polysaccharide usually has a shape protruding from the lipid of the cell membrane and functions mainly for defense from the host. Its sugar chain structure is extremely diverse and varies depending on the type of fungus. On the other hand, there are many common structures, and the sugar that first binds to lipids in the root part, that is, the cell membrane, is usually glucose.
  • the commonality of enzymes and genes involved in the synthesis of capsular polysaccharide is very high.
  • the commonality and specificity of the capsular gene for pathogens such as Streptococcus, Pseudomonas, and Staphylococcus are examined in detail.
  • the inventors have succeeded in identifying a gene that is particularly preferable as a detection target (target), that is, a gene having extremely high commonality.
  • the gene was shown to be extremely specific.
  • a method for detecting a pathogenic bacterium in a specimen comprising performing a nucleic acid amplification reaction targeting the capsular polysaccharide gene of the pathogenic bacterium, and determining the presence or absence of the pathogenic bacterium based on the amplification result.
  • the nucleic acid amplification reaction is a PCR method.
  • [3] Two or more types of pathogens selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella The method according to [2], wherein the PCR method is a multiplex PCR method.
  • the capsular polysaccharide gene is a cpsE gene for Streptococcus, a capE gene for Staphylococcus, a kpsF gene for Clostridium, a wzz gene for Pseudomonas, and pathogenic
  • E E.
  • a method for simultaneously detecting a plurality of types of pathogenic bacteria including the following steps (1) to (4): (1) preparing a DNA sample; (2) a step of preparing a plurality of primer sets, wherein each primer set is a primer set targeting capsular polysaccharide genes of different target pathogens; (3) performing a nucleic acid amplification reaction using the DNA sample as a template and simultaneously using the plurality of sets of primers; (4) A step of detecting an amplification product.
  • the method according to [5], wherein the nucleic acid amplification reaction is a multiplex PCR method.
  • the target pathogen is selected from two or more selected from Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic Escherichia coli, and Salmonella
  • the method according to [5] or [6], comprising a pathogen comprising a pathogen.
  • a primer set targeting the cpsE gene of Streptococcus, a primer set targeting the capE gene of Staphylococcus, a primer set targeting the kpsF gene of Clostridium, A primer set targeting the wzz gene of Pseudomonas, a primer set targeting the wcaA gene of pathogenic Escherichia coli, a primer set targeting the wbaP gene of Salmonella, and the rfc gene of Salmonella A kit for detecting pathogens, comprising two or more primer sets selected from the group consisting of primer sets targeted at.
  • capsular polysaccharide (schematic diagram) of pathogenic bacteria and structure of capsular polysaccharide gene.
  • the leftmost lane is a molecular weight marker.
  • the target amplification product is the band indicated by the arrow.
  • the rightmost lane is a molecular weight marker.
  • the target amplification product is the band indicated by the arrow.
  • Streptococcus agalactie genomic DNA and Streptococcus anjinosas genomic DNA were used as template DNA.
  • the band indicated by the arrow (upper: Streptococcus anjinosas, lower: Streptococcus agalactie) is the target amplification product.
  • On the left is the result of DNA amplification from genomic DNA using primers designed to target the capE gene (381 bp) of Staphylococcus aureus.
  • the rightmost lane is a molecular weight marker.
  • the target amplification product is the band indicated by the arrow.
  • On the right is the result of DNA amplification from genomic DNA using a primer set designed to target the rfc gene (311 bp) of Salmonella enterica.
  • the leftmost lane is a molecular weight marker.
  • the target amplification product is the band indicated by the arrow.
  • the template DNA used was Staphylococcus aureus genomic DNA and Salmonella enterica genomic DNA. A band corresponding to each target gene is observed.
  • the first aspect of the present invention relates to a method for detecting pathogenic bacteria.
  • the detection method of the present invention is characterized in that a nucleic acid amplification reaction targeting a capsular polysaccharide gene of a pathogenic bacterium is performed. That is, in the detection method of the present invention, a nucleic acid amplification reaction targeting the capsular polysaccharide gene of a pathogenic bacterium is performed, and the presence or absence of the pathogenic bacterium is determined based on the amplification result. When a specific amplification product is recognized, the target pathogen is detected.
  • nucleic acid amplification reactions examples include PCR (Polymerase chain reaction) method or its modification, LAMP (Loop-Mediated Isothermal Amplification) method (Tsugunori Notomi et al. Nucleic Acids Research, Vol.28, No.12, e63, 2000; Kentaro Nagamine, Keiko Watanabe et al. Clinical Chemistry, Vol. 47, No. 9, 1742-1743, 2001), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids (Patent No. 3433929, Patent No.
  • NASBA Nucleic Acid Sequence-Based Amplification
  • LCR Liigase Chain Reaction
  • 3SR Self-sustained Sequence Replication
  • SDA Standard Displacement Amplification
  • TMA Transcription Mediated Amplification
  • RCA Rolling Circle Amplification
  • a capsular polysaccharide gene that has been found to be highly common among pathogens of the same genus is targeted for detection.
  • the “target of detection” is also called “target” or “detection probe” in accordance with the conventional practice.
  • a highly common one is a chain length determining gene present on the 5 ′ side of the operon, glucose that synthesizes the first sugar of the sugar chain unit, galactose, Alternatively, it was a N-acetylglucosamine transferase gene, a flippase gene that transfers a sugar chain unit to the outside of the cell membrane.
  • the commonality of the chain length determining gene and the glucose, galactose, or N-acetylglucosamine transferase gene among the pathogenic bacteria is high. Therefore, the chain length determining gene or the first glycosyltransferase gene is preferably targeted.
  • pathogenic bacteria to be detected is not particularly limited.
  • target pathogens include Streptococcus, Staphylococcus, Clostridium, Pseudomonas, pathogenic E. coli (enteropathogenic E. coli (EPEC), intestinal tissue invasive E. coli (EIEC) ), Enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), intestinal agglutinating E. coli (EAggEC)), and Salmonella.
  • EEC enteropathogenic E. coli
  • ETEC enterohemorrhagic Escherichia coli
  • EAggEC intestinal agglutinating E. coli
  • a nucleic acid amplification reaction capable of simultaneously amplifying a plurality of targets, such as multiplex PCR, two or more, preferably three or more, more preferably four of the pathogenic bacteria exemplified here.
  • targets such as multiplex PCR
  • the above is the target pathogen.
  • the present inventors have succeeded in identifying genes with extremely high commonality and specificity for each genus for a plurality of types of pathogenic bacteria.
  • the specified gene is targeted and the detection specificity and accuracy are improved.
  • the gene which becomes a target in the said aspect is shown for every kind of pathogenic microbe (Table 1).
  • sequence of the target gene of the genus Streptococcus (cpsE gene)
  • sequence of S. anginosus is SEQ ID NO: 1
  • sequence of S. gordonii is SEQ ID NO: 2
  • sequence of S. oralis The sequence is shown in SEQ ID NO: 3
  • sequence of S. pneumoniae is shown in SEQ ID NO: 4
  • sequence of S. agalactiae is shown in SEQ ID NO: 5, respectively.
  • sequence of S. aureus is shown in SEQ ID NO: 6.
  • a Clostridial target gene (kpsF gene)
  • the sequence of Tetani (C. ⁇ tetani) is shown in SEQ ID NO: 7.
  • a target gene of the genus Pseudomonas (wzz gene)
  • the sequence of aeruginosa (P. ⁇ aeruginosa) is shown in SEQ ID NO: 8.
  • An example of the sequence of the target gene (wcaA gene) of pathogenic E. coli is shown in SEQ ID NO: 9.
  • Examples of sequences of Salmonella target genes (wbaP gene, rfc gene) are shown in SEQ ID NO: 10 (wbaP gene) and SEQ ID NO: 23 (rfc gene).
  • a nucleic acid amplification reaction using one to several primer sets (for example, PCR with a single primer set, multiple primer sets) In the case of a set, multiplex PCR) will be performed.
  • a nucleic acid amplification reaction for example, multiplex PCR
  • a nucleic acid amplification reaction may be carried out using a primer set (one to several sets for each genera) prepared for each genera.
  • Primer set for Streptococcus (designed based on the sequence of the cpsIaE gene of Streptococcus agaractie) Direct: 5'-CAATCAAATGACAGGGCTAAT-3 '(SEQ ID NO: 11) Reverse: 5'-TAAAACTAAGGCGTCGCTT-3 '(SEQ ID NO: 12)
  • Primer set for Streptococcus genus (designed based on the sequence of Streptococcus anjinosas cpsE gene) Direct: 5'-TGTACGTAGATGCCGAGG-3 '(SEQ ID NO: 13) Reverse: 5′-TTAAAGCTCAATCGCCGC-3 ′ (SEQ ID NO: 14)
  • Primer set for Pseudomonas (designed based on the sequence of Pseudomonas aeruginosa wzz gene) Direct: 5'-CGTGAAGATC
  • Each oligonucleotide constituting the primer set may be prepared by a conventional method. For example, it can be chemically synthesized using a general-purpose DNA synthesizer.
  • the detection method of the present invention typically uses a PCR method. This improves versatility and quickness.
  • a multiplex PCR method is employed.
  • the multiplex PCR method is a PCR method using a plurality of primer sets at the same time, and enables detection of a plurality of targets at once (for example, US Pat. No. 5,582,989, Journal of Microbiological Methods 68 (2007) 52 ⁇ (See 59). Therefore, if the multiplex PCR method is used, the types of pathogenic bacteria that can be detected at a time are increased, and the rapidity and convenience are further improved.
  • the following steps (1) to (4) are performed to detect a plurality of types of pathogenic bacteria simultaneously.
  • Step of preparing a DNA sample (2) Step of preparing a plurality of primer sets, each primer set being a primer set targeting capsular polysaccharide genes of different target pathogens (3) A step of performing a nucleic acid amplification reaction using the DNA sample as a template and simultaneously with the plurality of sets of primers (4) A step of detecting an amplification product
  • step (1) prepare a DNA sample.
  • the detection method of the present invention can be used to detect pathogenic bacteria in various test materials (specimens). Examples of test materials include soil, sewage, food, wiped samples, urine, stool, urine storage, sputum, and vomit. After removing impurities or diluting as necessary, a DNA sample is prepared from the test material, and the detection method of the present invention is applied.
  • a DNA sample may be prepared by a conventional method. Many kits for preparing DNA samples are also commercially available, and DNA samples can be easily obtained by using such kits.
  • each primer set has a different target pathogenic capsular polysaccharide.
  • the number of primer sets is, for example, 2 to 5 sets. As the number of primer sets increases, the number of pathogenic bacteria that can be detected simultaneously increases, but there is a problem that the possibility of non-specific amplification increases and the design of primers becomes difficult. Therefore, the number of primer sets is preferably 5 to 30 sets.
  • the number of primer sets prepared for one genus of pathogenic bacteria may not be one. That is, two or more primer sets (for example, 2, 3, 4, or 5) may be prepared for one genus.
  • a primer set labeled with a labeling substance may be used.
  • labeling substances are fluorescent substances, chemiluminescent substances, biotin, and radioisotopes.
  • step (3) the nucleic acid amplification reaction is performed using the DNA sample prepared in step (1) as a template and using the plurality of primer sets prepared in step (2) simultaneously.
  • multiplex PCR is performed.
  • Each step of the nucleic acid amplification reaction may be performed by a conventional method. If PCR is employed, for example, a commercially available PCR device (for example, thermal cycler personal manufactured by Takara Bio Inc.) can be used. Numerous PCR kits that include enzymes, reagents, etc. are also commercially available. By using such a kit, each step of PCR can be carried out easily. PCR conditions may be set in consideration of primer Tm and the like. Examples of PCR conditions are as follows.
  • heat denaturation is 90 ° C to 98 ° C
  • annealing is 30 ° C to 65 ° C
  • extension reaction is 65 ° C to 75 ° C.
  • the number of reaction cycle repetitions is, for example, 20-40.
  • Ex Taq registered trademark, Takara Bio Inc.
  • gene taq Nippon Gene
  • KOD plus Toyobo Co., Ltd.
  • the amplification product is detected, but the detection method is not particularly limited.
  • a method using electrophoresis a method using chromatography, a method using a DNA array, and the like can be employed.
  • a specific example of the method using electrophoresis is as follows. The reaction solution after PCR is applied to a gel such as agarose and subjected to electrophoresis, followed by staining with ethidium bromide. The presence or absence of the target DNA fragment (amplified product) is determined using the position (movement distance) of the band that appears by staining as an index. In addition, the amount of the target DNA fragment may be determined based on the density of the band.
  • 2% TAE Tris acetate, EDTA
  • TBE Tris borate, EDTA
  • the detection kit of the present invention is a primer set targeting the Streptococcus cpsE gene, a primer set targeting the Staphylococcus capE gene, and Clostridium spp.
  • Primer set targeting kpsF gene, primer set targeting Pseudomonas wzz gene, primer set targeting wcaA gene of pathogenic E. coli, primer set targeting wbaP gene of Salmonella, and rfc of Salmonella It includes two or more primer sets selected from the group consisting of primer sets targeting genes.
  • primer sets may be used for one genus of pathogenic bacteria.
  • An example of this embodiment an example of a kit capable of detecting Streptococcus spp. And Pseudomonas spp.
  • a primer set targeting the Streptococcus cpsE gene the cpsE gene of a specific species (one or more) And a primer set targeting a cpsE gene of one or more species different from the species, and a primer set targeting a Pseudomonas wzz gene as a specific species
  • a kit comprising a primer set targeting one or more wzz genes and a primer set targeting a wzz gene of a species (one or more) different from the species.
  • two primer sets are used for one genus of pathogenic bacteria, but the present invention is not limited to this.
  • the primer constituting the primer set is not particularly limited as long as it can specifically amplify the target gene or a part thereof.
  • the length of the primer is not limited as long as it functions as a primer (for example, a PCR primer) in the nucleic acid amplification reaction employed, and is, for example, 15 to 30 bp, preferably 20 to 30 bp, more preferably 20 to 25 bp. There may be 1 to several, preferably 1 to 5, more preferably about 1 to 3 mismatches between the primer and the region of the target gene to which it hybridizes.
  • primer set examples include the above-mentioned primer set for the genus Streptococcus (SEQ ID Nos. 11 and 12: designed based on the sequence of the cpsIaE gene of Streptococcus agalactie), and the primer set for the genus Streptococcus (SEQ ID Nos.
  • Reagents necessary for each step of nucleic acid amplification reaction typically PCR (DNA polymerase, buffer solution, etc.), reagents required for detection (reagents for gel preparation, stain solution, etc.), containers, instruments, etc. It may be included in the detection kit of the present invention. Usually, an instruction manual is attached to the detection kit of the present invention.
  • Capsular polysaccharides are present in almost all pathogens and are closely related to pathogenicity (Fig. 1).
  • the capsular polysaccharide usually has a shape protruding from the lipid of the cell membrane and functions mainly for defense from the host. Its sugar chain structure is extremely diverse and varies depending on the type of fungus. On the other hand, there are many common structures, and the sugar that first binds to the lipids of the cell membrane, ie, the cell membrane, is usually glucose or galactose.
  • the commonality of enzymes and genes involved in the synthesis of capsular polysaccharide is very high.
  • cpsE gene for Streptococcus pathogens As mentioned above, cpsE gene for Streptococcus pathogens, wzz gene for Pseudomonas pathogens, capE gene for Staphylococcus pathogens, kpsF gene for Clostridium pathogens, pathogenicity
  • the wcaA gene was found for E. coli and the wbaP and rfc genes were found for Salmonella.
  • a detection primer set was designed for each target gene as follows. (Primer set targeting the cpsIaE gene of Streptococcus agalactie) Direct: 5'-CAATCAAATGACAGGGCTAAT-3 '(SEQ ID NO: 11) Reverse: 5'-TAAAACTAAGGCGTCGCTT-3 '(SEQ ID NO: 12) (Primer set targeting the cpsE gene of Streptococcus anginosas) Direct: 5'-TGTACGTAGATGCCGAGG-3 '(SEQ ID NO: 13) Reverse: 5′-TTAAAGCTCAATCGCCGC-3 ′ (SEQ ID NO: 14) (Primer set targeting the wzz gene of Pseudomonas aeruginosa) Direct: 5'-CGTGAAGATCGTAT-3 '(SEQ ID NO: 15) Reverse: 5'-GGAATAAAAGGATCATC-3 '(SEQ ID NO: 16) (Primer set
  • test groups negative control (no DNA sample), soil DNA sample (200 ng) only, soil DNA sample (200 ng) and Streptococcus agalactiae genomic DNA (1 fg: 2x10 -25 mol), soil DNA sample (200 ng) ) And Streptococcus agalactie genomic DNA (1 pg: 2x10 -22 mol), soil DNA sample (200 pg) and Streptococcus agalactia genomic DNA (1 ng: 2x10 -19 mol), and the above primer set (SEQ ID NO: 11) And SEQ ID NO: 12).
  • KOD plus DNA polymerase was used and the PCR conditions were as follows. ⁇ PCR conditions> 2 minutes at 96 ° C, 10 seconds at 98 ° C; 30 seconds at 50 ° C; 20 seconds at 68 ° C (35 cycles)
  • test groups negative control (no DNA sample), soil DNA sample (200 ng) only, soil DNA sample (200 ng) and Pseudomonas aeruginosa genomic DNA (1 fg), soil DNA sample (200 ng) and Pseudomonas aeruginosa Set genomic DNA (1 fg), soil DNA sample (200 pg) and Pseudomonas aeruginosa genomic DNA (1 ng), soil DNA sample (200 pg) and Pseudomonas aeruginosa genomic DNA (1 ⁇ g), and set the above primer set (sequence) PCR was performed using No. 15 and SEQ ID No. 16).
  • Each sample after PCR was subjected to electrophoresis (2% TAE (Tris acetic acid, EDTA) agarose) to confirm the presence or absence of amplification. As shown in the left of FIG. 5, specific amplification of the target gene was observed. It was also shown that it can be detected with high sensitivity from a small amount of sample. As described above, the high specificity of the primer was confirmed.
  • TAE Tris acetic acid, EDTA
  • bands corresponding to each primer set were recognized. That is, it was shown that two target genes can be amplified and detected simultaneously by multiplex PCR.
  • pathogenic bacteria in a specimen are detected by targeting a capsular polysaccharide gene that is highly common and closely related to pathogenicity.
  • a PCR method that is versatile and excellent in rapidity is used.
  • pathogenic bacteria can be detected from the environment simply, quickly and specifically.
  • the present invention exhibits high specificity for pathogenic bacteria.
  • the detection method of the present invention is suitable for simultaneously detecting a plurality of pathogenic bacteria that may be present in samples such as soil, sewage, and food.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de détection d'une bactérie pathogène dont on suspecte la présence dans un échantillon, de manière simple, rapide et spécifique. L'invention concerne également une réaction d'amplification d'un acide nucléique qui cible un gène de polysaccharide capsulaire de la bactérie pathogène. La présence ou l'absence de la bactérie pathogène dans l'échantillon est déterminée en se basant sur les résultats de l'amplification.
PCT/JP2010/060940 2009-07-08 2010-06-28 Procédé de détection de bactéries pathogènes, et kit d'utilisation de ce procédé WO2011004725A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009161351 2009-07-08
JP2009-161351 2009-07-08

Publications (1)

Publication Number Publication Date
WO2011004725A1 true WO2011004725A1 (fr) 2011-01-13

Family

ID=43429146

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/060940 WO2011004725A1 (fr) 2009-07-08 2010-06-28 Procédé de détection de bactéries pathogènes, et kit d'utilisation de ce procédé

Country Status (1)

Country Link
WO (1) WO2011004725A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5948900A (en) * 1994-05-16 1999-09-07 Uab Research Foundation Streptococcus pneumoniae capsular polysaccharide genes and flanking regions
US6183973B1 (en) * 1997-06-19 2001-02-06 Umbi - University Of Maryland Biotechnology Institute Vibrio vulnificus molecular probes, antibodies, and proteins
WO2005064016A1 (fr) * 2003-12-26 2005-07-14 Prima Meat Packers, Ltd. Procédé de détection multiplex de micro-organismes
JP2007274934A (ja) * 2006-04-04 2007-10-25 Nippon Meat Packers Inc プライマーセット及び食中毒細菌の検出方法
JP2009039046A (ja) * 2007-08-09 2009-02-26 Yamaguchi Univ 肺炎原因菌検出用プライマーセット

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5948900A (en) * 1994-05-16 1999-09-07 Uab Research Foundation Streptococcus pneumoniae capsular polysaccharide genes and flanking regions
US6183973B1 (en) * 1997-06-19 2001-02-06 Umbi - University Of Maryland Biotechnology Institute Vibrio vulnificus molecular probes, antibodies, and proteins
WO2005064016A1 (fr) * 2003-12-26 2005-07-14 Prima Meat Packers, Ltd. Procédé de détection multiplex de micro-organismes
JP2007274934A (ja) * 2006-04-04 2007-10-25 Nippon Meat Packers Inc プライマーセット及び食中毒細菌の検出方法
JP2009039046A (ja) * 2007-08-09 2009-02-26 Yamaguchi Univ 肺炎原因菌検出用プライマーセット

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
COLLINS L.V.: "Molecular Cloning, Characterization, and Nucleotide Sequence of the rfc Gene, Which Encodes an O-Antigen Polymerase of Salmonella typhimurium", JOURNAL OF BACTERIOLOGY, vol. 173, no. 8, 1991, pages 2521 - 2529 *
DATABASE GENBANK [online] 13 June 2002 (2002-06-13), RAYMOND C.K. ET AL.: "Definition: Pseudomonas aeruginosa serotype 020 putative O-antigen biosynthesis gene cluster, partial sequence.", retrieved from http://www. ncbi.nlm.nih.gov/nuccore/20559984 Database accession no. AF498413 *
DATABASE GENBANK [online] 13 March 1997 (1997-03-13), SAU S. ET AL.: "Definition:Staphylococcus aureus type 8 capsule genes, cap8A, cap8B, cap8C, cap8D, cap8E, cap8F, cap8G, cap8H, cap8I, cap8J, cap8K, cap8L, cap8M, cap8N, cap80, cap8P, complete cds.", retrieved from http://www.ncbi.nlm.nih.gov/ nuccore/1657639 Database accession no. U73374 *
DATABASE GENBANK [online] 16 May 2001 (2001-05-16), HOBBS M. ET AL.: "Definition: putative glycosyl transferase [Escherichia coli].", retrieved from http://www.ncbi.nlm.nih. gov/protein/1407606 Database accession no. AAC77836 *
DATABASE GENBANK [online] 20 July 2008 (2008-07-20), BRUGGEMANN H. ET AL.: "Definition: polysialic acid capsule expression protein kpsF [Clostridium tetani E88].", retrieved from http://www.ncbi.nlm.nih. gov/sviewer/viewer.fcgi?val=28210619&sat=NCBI& satkey=23381576 Database accession no. NP_781563 *
DATABASE GENBANK [online] 29 April 2005 (2005-04-29), CIESLEWICZ M.J. ET AL.: "Definition: Streptococcus agalactiae strain 7271 Cps7C, Cps7D, Cps7E, Cps7F, Cps7G, Cps7H, Cps7M, Cps7I, Cps7J, Cps7K, Cps7L, NeuB, NeuC, NeuD, and NeuA genes, complete cds.", retrieved from http://www.ncbi.nlm.nih. gov/nuccore/38640633 Database accession no. AY376403 *
DATABASE GENBANK [online] 9 September 2004 (2004-09-09), BROWN P.K. ET AL.: "Definition: S.enterica rfbJ gene cluster.", retrieved from http://www.ncbi.nlm.nih.gov/nuccore/ 47004 Database accession no. X61917 *
HIROYUKI TSUNASHIMA ET AL.: "Byogenkin Yurai no Kyomaku Tato Seigosei Idenshigun no Tanri to Kaiseki", ABSTRACTS OF THE 74TH ANNUAL MEETING (YOKOHAMA) OF THE SOCIETY OF CHEMICAL ENGINEERS, vol. 74, 18 February 2009 (2009-02-18), pages S303 *

Similar Documents

Publication Publication Date Title
Millar et al. Molecular diagnostics of medically importantbacterial infections
Handschur et al. Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood
Huang et al. Quadruplex real-time PCR assay for detection and identification of Vibrio cholerae O1 and O139 strains and determination of their toxigenic potential
Dreier et al. Real-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products
Volokhov et al. Sequencing of the intergenic 16S-23S rRNA spacer (ITS) region of Mollicutes species and their identification using microarray-based assay and DNA sequencing
JP2013520186A (ja) 緑膿菌の血清型決定のためのアッセイ法およびキット、ならびにそのような方法およびキットにおいて有用なオリゴヌクレオチド配列
Andersen et al. Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material
Sławiak et al. Multiplex detection and identification of bacterial pathogens causing potato blackleg and soft rot in Europe, using padlock probes
US20160060684A1 (en) Rapid salmonella serotyping assay
JP2007274934A (ja) プライマーセット及び食中毒細菌の検出方法
Gracias et al. Nucleic acid sequence‐based amplification (NASBA) in molecular bacteriology: a procedural guide
Kim et al. Simultaneous identification of 13 foodborne pathogens by using capillary electrophoresis–single strand conformation polymorphism coupled with multiplex ligation-dependent probe amplification and its application in foods
EP2347013B1 (fr) Procédé pour la détection spécifique d'espèces d'arn de faible abondance dans un échantillon biologique
US9932642B2 (en) Rapid Salmonella serotyping assay
Kim et al. Rapid and simple detection of the invA gene in Salmonella spp. by isothermal target and probe amplification (iTPA)
KR20030077790A (ko) 병원성 미생물의 유전자 증폭용 프라이머, 이를 이용한병원성 미생물의 검출방법, 및 검출키트
Amann et al. Typing in situ with probes
WO2011004725A1 (fr) Procédé de détection de bactéries pathogènes, et kit d'utilisation de ce procédé
CN105256041B (zh) 对亲水气单胞菌o44，o24，o25和o28特异的核苷酸及应用
JP2022025153A (ja) ビフィドバクテリウム・ブレーベに対して特異的な１以上のプライマーまたはプローブ
KR20010072357A (ko) 세균 검출용 유전자 및 이를 이용한 세균의 검출방법
KR102009326B1 (ko) cpa와 cpe 타겟 유전자를 통해 클로스트리디움 퍼프린젠스를 검출할 수 있는 Singleplex Real-Time PCR 키트 개발
STAMATOSkI et al. Optimized genotyping method for identification of bacterial contaminants in pharmaceutical industry
KR101332366B1 (ko) 미생물 농약균주 바실러스 튜린겐시스 및 식중독균 바실러스 세레우스 구별용 프라이머 셋트 및 이를 이용한 구별 방법
Kouguchi et al. Sequence-specific detection using a universal probe system based on the formation of a four-way junction structure

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10797036

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10797036

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP