WO2019221219A1 - Procédé d'examen d'une bactérie, micropuce pour l'examen d'une bactérie, kit d'examen de bactérie, ensemble des sondes pour l'examen d'une bactérie et ensemble d'amorces pour l'examen d'une bactérie - Google Patents

Procédé d'examen d'une bactérie, micropuce pour l'examen d'une bactérie, kit d'examen de bactérie, ensemble des sondes pour l'examen d'une bactérie et ensemble d'amorces pour l'examen d'une bactérie Download PDF

Info

Publication number
WO2019221219A1
WO2019221219A1 PCT/JP2019/019446 JP2019019446W WO2019221219A1 WO 2019221219 A1 WO2019221219 A1 WO 2019221219A1 JP 2019019446 W JP2019019446 W JP 2019019446W WO 2019221219 A1 WO2019221219 A1 WO 2019221219A1
Authority
WO
WIPO (PCT)
Prior art keywords
listeria
bacteria
seq
probes
nos
Prior art date
Application number
PCT/JP2019/019446
Other languages
English (en)
Japanese (ja)
Inventor
隆明 山崎
伸哉 寿命
淳憲 一色
Original Assignee
東洋製罐グループホールディングス株式会社
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
Priority claimed from JP2018190417A external-priority patent/JP2019201626A/ja
Application filed by 東洋製罐グループホールディングス株式会社 filed Critical 東洋製罐グループホールディングス株式会社
Publication of WO2019221219A1 publication Critical patent/WO2019221219A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the present invention relates to an inspection technique for Listeria spp. Which is regarded as a problem in food manufacturing facilities.
  • Listeria monocytogenes is a pathogenic species of food poisoning.
  • Listeria Monocytogenes-induced food poisoning is likely to occur in pregnant women, newborns, the elderly, and people with weakened immune systems. In the United States, about 1,600 people have been affected each year in the past, and about 260 deaths have occurred. It has been reported that
  • Non-Patent Document 1 the gene region to be examined is iap, Listeria grayi, Listeria innocua, Listeria ivanovii, Listeria monocytogenes, Listeria monocytogenes, Listeria monocytogenes. (Listeria seeligeri), the method of detecting 6 microbial species of Listeria welshimeri is described.
  • Non-Patent Document 2 describes a technique for detecting the same six bacterial species as described above using the gene region to be examined as an IGS. However, none of these documents describes a technique for detecting all 17 species of Listeria.
  • Listeria floridensis Listeria weihenstephanensis, Listeria cornelensis, Listeria aria, Listeria aria, Listeria aria aria.
  • the present invention has been made in view of the above circumstances, and is capable of accurately identifying all 17 bacterial species in the genus Listeria, a microarray for testing bacteria, a kit for testing bacteria, An object is to provide a probe set for testing and a primer set for testing bacteria.
  • a method for examining bacteria is a method for examining the presence of bacteria in a sample
  • the target bacteria are Listeria belonging to the following (1) to (10): (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria boriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria ⁇ ⁇ Georgiani ( Listeria yakmannii), (10) Listery Listeria marthii
  • the method includes (a) a preparation step of preparing a reaction solution containing a primer set for amplification of an IGS (intergenic spacer) region of the rRNA gene, and (b) a reaction obtained by the preparation step
  • the method includes an amplification step of amplifying the nucleic acid of the target bacteria contained in the sample using a liquid.
  • the microarray for testing bacteria of the present invention is a microarray for testing bacteria that detects the presence of bacteria in a sample, and is an IGS of the rRNA gene of Listeria belonging to the above (1) to (10). At least one of the probes having the base sequences shown in SEQ ID NOs: 32-43 and 47 selected from the (intergenic spacer) region is immobilized.
  • the bacterial test kit of the present invention is a bacterial test kit for detecting the presence of bacteria in a sample, and the IGS of the Listeria spp. RRNA gene described in (1) to (10) above.
  • a primer set consisting of primers having the base sequences shown in SEQ ID NOs: 16 and 17.
  • the bacterial test probe set of the present invention is a bacterial test probe set for testing the presence of bacteria in a sample, and the rRNA gene of Listeria belonging to the above (1) to (10)
  • the IGS (intergenic spacer) region is selected from probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47.
  • the bacterial test primer set of the present invention is a bacterial test primer set for testing the presence of bacteria in a sample, the rRNA gene of Listeria belonging to the above (1) to (10) From primers of the nucleotide sequences shown in SEQ ID NOs: 16 and 17 for amplifying a nucleic acid that hybridizes with at least one of the probes of the nucleotide sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the IGS (intergenic spacer) region of The configuration is as follows.
  • FIG. 1 it is a figure which shows the base sequence of the primer used in order to analyze the base sequence of the IGS area
  • the IGS region of various Listeria species Listeria floridensis, Listeria wehenstephanensis, Listeria cornelensis, Listeria liparia, Listeria grandensis
  • Samples used in Test 1 for the method for examining bacteria according to the embodiment of the present invention (Listeria Locotier, Listeria Siligelli, Listeria Wersimeli, Listeria Floridensis, Listeria Wehenstephanensis, Listeria Cornelensis, Listeria Liparia, Listeria Grandensis, It is a figure which shows the sample number of the Listeria boriae, Listeria New Yorkensis, Listeria Aquatica, Listeria freshmanni, Listeria marti), and a test strain. It is a figure which shows the base sequence of the primer for amplifying the plasmid as an internal control used by each test, such as the inspection method of bacteria concerning embodiment of this invention, and its amplification object DNA.
  • FIG. 6 is a diagram showing the detection results (sample numbers 1 to 14) of test 1 using probes (SEQ ID NOs: 18 to 31) used in the method for examining bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing the detection results (sample numbers 15 to 28) of test 1 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the detection results (sample numbers 29 to 43) of test 1 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the detection results (sample numbers 1 to 14) of test 1 using probes (SEQ ID NOs: 32 to 45) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the detection results (sample numbers 15 to 28) of test 1 using probes (SEQ ID NOs: 32 to 45) used in the method for examining bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the detection results (sample numbers 29 to 43) of test 1 using probes (SEQ ID NOs: 32 to 45) used in the method for examining bacteria according to the embodiment of the present invention.
  • Sample numbers of samples (Staphylococcus aureus, Escherichia coli, Shigella, Nagvibrio, Salmonella) used in Test 2 for the test method of bacteria according to the embodiment of the present invention to confirm the presence or absence of false positives, and test It is a figure which shows a strain.
  • FIG. 7 is a diagram showing detection results (sample numbers 44 to 59) of test 2 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing the detection results (sample numbers 44 to 59) of test 2 using probes (SEQ ID NOs: 32 to 45) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the presence of a test strain, DNA concentration, and internal control (IC) added to a sample (sample numbers 60 to 65) used in Test 3 for a method for examining bacteria according to an embodiment of the present invention. is there.
  • FIG. 4 is a diagram showing the presence of a test strain, a DNA concentration, and an internal control (IC) added to a sample (sample numbers 66 to 71) used in Test 3 for a bacterial inspection method and the like according to an embodiment of the present invention. is there.
  • FIG. 7 is a diagram showing detection results (sample numbers 60 to 71) of test 3 using probes (SEQ ID NOs: 18 to 31) used in the method for examining bacteria according to the embodiment of the present invention.
  • FIG. 7 is a diagram showing detection results (sample numbers 60 to 71) of test 3 using probes (SEQ ID NOs: 32 to 45) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing the detection results (sample numbers 1a to 14a) of test 4 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 5 is a diagram showing detection results (sample numbers 15a to 28a) of test 4 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 7 is a diagram showing detection results (sample numbers 29a to 43a) of test 4 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing the detection results (sample numbers 1a to 14a) of test 4 using probes (SEQ ID NOs: 32 to 45, 47) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 7 is a diagram showing detection results (sample numbers 15a to 28a) of test 4 using probes (SEQ ID NOs: 32 to 45, 47) used in the method for examining bacteria according to the embodiment of the present invention.
  • FIG. 4 is a diagram showing detection results (sample numbers 29a to 43a) of test 4 using probes (SEQ ID NOs: 32 to 45, 47) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing detection results (sample numbers 44a to 59a) of test 5 using probes (SEQ ID NOs: 18 to 31) used in the method for testing bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing detection results (sample numbers 44a to 59a) of test 5 using probes (SEQ ID NOs: 32 to 45 and 47) used in the method for examining bacteria according to the embodiment of the present invention.
  • FIG. 6 is a diagram showing the detection results (sample numbers 63a, 65a, 72a, 69a, 71a, 73a) of test 6 using probes (SEQ ID NOs: 32-45, 47) used in the method for testing bacteria according to the embodiment of the present invention. is there.
  • the method for examining bacteria is a method for examining the presence of bacteria in a sample, and the target bacteria is selected from the Listeria species described in (1) to (10) below, (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria yakmannii, (10) Listeria marthii,
  • This method (A) a preparation step of preparing a reaction solution containing a primer set for amplification of an IGS (intergenic spacer) region of the rRNA gene; and (B) An amplification step of amplifying the nucleic acid of the target bacteria contained in the sample using the reaction solution obtained in the preparation step is characterized.
  • the target bacteria further include bacteria selected from Listeria spp. Described in (11) to (17) below.
  • (11) Listeria grayi (12) Listeria innocua (13) Listeria ivanovii (14) Listeria monocytogenes (15) Listeria rocourtiae (16) Listeria seeligeri (17) Listeria welshimeri
  • IGS intergenic spacer
  • F / R indicates that F is a forward primer and R is a reverse primer.
  • FIG. 2 shows the base sequences in the IGS region of Listeria floridensis, Listeria allegedlystephanensis, Listeria cornelensis, Listeria liparia, and Listeria grandensis (SEQ ID NOs: 3 to 7 in order).
  • FIG. 3 shows the base sequences in the IGS regions of Listeria Booliae, Listeria Nuyoenensis, Listeria Aquatica, Listeria Freshmanni, and Listeria Marti (sequence numbers 8 to 12 in order).
  • the IGS region was analyzed again in order to find a more effective probe, and the base sequence (SEQ ID NO: 46) shown in FIG. 23 was obtained.
  • the nucleotide sequences in the IGS regions of Listeria Gray, Listeria Inocure, Listeria Ivanobi, Listeria Monocytogenes, Listeria Locoutier, Listeria Shiligeri, and Listeria Wersimeli were obtained from DDBJ. Then, by comparing the base sequences in the IGS region of these 17 kinds of Listeria species, the design location of the bacterial test probe set and bacterial test primer set of this embodiment was determined.
  • the primer set for amplification in the IGS (intergenic spacer) region of the rRNA gene contained in the reaction solution prepared in the above preparation step is a forward sequence consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 16 in FIG. It consists of a primer and a reverse primer consisting of an oligonucleotide having the base sequence shown in SEQ ID NO: 17.
  • a probe set selected from the IGS (intergenic spacer) region of the rRNA gene that hybridizes with the nucleic acid amplified by the amplification step described above is based on the oligonucleotides of the nucleotide sequences shown in SEQ ID NOs: 18 to 31 in FIG. And at least one selected from the group consisting of probes consisting of oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 32-43 in FIG. Note that a probe for detecting a plasmid as an internal control used for determining whether or not DNA amplification by PCR reaction has been properly performed is shown in SEQ ID NO: 45 in FIG.
  • a probe having a nucleotide sequence shown in SEQ ID NO: 47 of FIG. 23 was designed as a more effective probe. That is, the probe set selected from the IGS (intergenic spacer) region of the rRNA gene that hybridizes with the nucleic acid amplified by the amplification step described above is a probe comprising an oligonucleotide having the nucleotide sequence shown in SEQ ID NOs: 18 to 31. It is also preferable to consist of at least one selected from the group consisting of probes consisting of oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 32 to 43 and 47.
  • the above reaction solution contains genomic DNA extracted from Listeria cells.
  • Genomic DNA can be extracted by a general method such as a method using a kit or a method using a DNA extraction apparatus.
  • the target region in the extracted genomic DNA is amplified. That is, a DNA fragment containing the target region in genomic DNA is amplified.
  • the method for amplifying the target region is not particularly limited, but the PCR method can be suitably used. A general thermal cycler or the like can be used as the PCR apparatus.
  • the target region in the genomic DNA can be suitably amplified by performing PCR under the following reaction conditions.
  • a solution having the following composition is preferably used. That is, a PCR reaction containing a nucleic acid synthesis substrate (dNTPmixture (dCTP, dATP, dTTP, dGTP)), primer set, nucleic acid synthase (such as HotStart DNA polymerase), sample genomic DNA, buffer solution, and sterilized water as the remaining components
  • dNTPmixture dCTP, dATP, dTTP, dGTP
  • primer set such as HotStart DNA polymerase
  • sample genomic DNA such as HotStart DNA polymerase
  • buffer solution such as HotStart DNA polymerase
  • hybridization can be performed as follows. That is, the amplification product obtained by the above amplification step is dropped onto the bacterial test microarray of this embodiment, and the label of the amplification product that has hybridized to the probe placed thereon is detected. Check for presence. Thereby, it is possible to identify Listeria belonging to the environment to be examined.
  • the detection of the label can be performed using a general label detection device such as a fluorescence scanning device.
  • a fluorescence scanning device For example, the fluorescence intensity of the amplification product is measured using the GENOGATE (R) reader of Toyo Seikan Group Engineering Co., Ltd. This can be done.
  • the measurement result is preferably obtained as an S / N ratio value (Signal to Noise ratio). This is because whether the measurement result is positive or negative can be determined with high accuracy based on the S / N ratio value. In the case of the microarray used in Examples described later, it can be determined as positive when the S / N ratio value is 3.0 or more.
  • the S / N ratio value described in the present specification is calculated by (median fluorescence intensity value ⁇ background value) ⁇ background value.
  • the label is not limited to fluorescence, and other labels can also be used.
  • the microarray for testing bacteria according to this embodiment is for testing the presence of bacteria in a sample, and an IGS (intergenic spacer) of the rRNA gene of Listeria belonging to the above (1) to (10) At least one of the probes having the base sequences shown in SEQ ID NOs: 32 to 43 and 47 selected from the region is immobilized.
  • the microarray for testing bacteria of this embodiment is further represented by SEQ ID NOs: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria belonging to the above (11) to (17). It is preferable that at least one of the probes of the base sequence is immobilized.
  • the microarray for testing bacteria of this embodiment can be obtained by immobilizing all or part of the probes having the base sequences shown in SEQ ID NOs: 18 to 43 and 47.
  • the microarray for testing bacteria can be produced by an existing general method using probes having the nucleotide sequences shown in SEQ ID NOs: 18 to 43 and 47.
  • an affixed DNA chip when produced as this microarray, it can be produced by immobilizing probes on a glass substrate with a DNA spotter and forming spots corresponding to each probe.
  • a synthetic DNA chip when produced, it can be produced by synthesizing a single-stranded oligo DNA having the above sequence on a glass substrate by a photolithography technique.
  • the substrate is not limited to glass, and a plastic substrate, a silicon wafer, or the like can also be used.
  • the shape of the substrate is not limited to a flat plate shape, and may be various three-dimensional shapes, and a substrate having a functional group introduced so that a chemical reaction can be performed on the surface can be used. .
  • test kit for bacteria of this embodiment is for testing the presence of bacteria in a sample, and an IGS (intergenic spacer) of the Listeria spp. RRNA gene described in (1) to (10) above.
  • IGS intergenic spacer
  • a primer set comprising primers of the base sequences shown in 16 and 17.
  • test kit for bacteria of the present embodiment is further represented by SEQ ID NOs: 18 to 31 selected from the IGS (intergenic spacer) region of the rRNA gene of Listeria belonging to the above (11) to (17). It is preferable that at least one of the probes of the base sequence is immobilized.
  • the probe set for testing bacteria is for testing the presence of bacteria in a sample.
  • the IGS (intergenic spacer) of the rRNA gene of Listeria belonging to the above (1) to (10) ) It is characterized by comprising at least one of the probes of the base sequences shown in SEQ ID NOs: 32 to 43, 47 selected from the region.
  • the bacterial probe set of the present embodiment further includes SEQ ID NOs: 18 to 31 selected from the IGS (intergenic spacer) region of the Listeria spp. RRNA gene described in (11) to (17) above. It is preferable to include at least one of the probes having the base sequences shown.
  • the bacterial test primer set of the present embodiment is for testing the presence of bacteria in a sample.
  • the probe set described above preferably includes at least one of the following probes (A) or (B), two or more of these, or all of them.
  • the probe set for testing bacteria of this embodiment has a length of 21 to 36 bases, and can be synthesized by a general DNA synthesizer.
  • the probes consisting of the nucleotide sequences shown in SEQ ID NOs: 18 to 43 and 47 used in Examples described later were all synthesized by a DNA synthesizer.
  • the bacterial test primer set of the present embodiment is also 19 to 22 bases in length, and the primers comprising the base sequences shown in SEQ ID NOs: 16 and 17 used in PCR in the examples were synthesized by a DNA synthesizer. We used what we did.
  • the primers composed of the nucleotide sequences shown in SEQ ID NOs: 1 and 2 used for PCR in the examples were also synthesized by a DNA synthesizer.
  • the probes consisting of the base sequences shown in SEQ ID NOs: 18 to 43, 47 in this embodiment are not limited to the sequences themselves, and one or several bases are deleted from the base sequences shown in SEQ ID NOs: 18 to 43, 47. Substituted or added probes can be used. Probes that can hybridize under stringent conditions to a nucleic acid fragment consisting of a base sequence complementary to the base sequences shown in SEQ ID NOs: 18 to 43 and 47 can also be used. In addition, these probes and probes having a base sequence complementary to the probes consisting of the base sequences shown in SEQ ID NOs: 18 to 43 and 47 can also be used.
  • the stringent condition refers to a condition where a specific hybrid is formed and a non-specific hybrid is not formed.
  • DNAs having high homology (homology is 90% or more, preferably 95% or more) to DNAs having the nucleotide sequences shown in SEQ ID NOs: 18 to 43 and 47 are shown in SEQ ID NOs: 18 to 43 and 47.
  • Conditions for hybridizing with DNA consisting of a base sequence and DNA each consisting of a complementary base sequence are mentioned. Usually, it means a case where hybridization occurs at a temperature about 5 ° C. to about 30 ° C., preferably about 10 ° C. to about 25 ° C. lower than the melting temperature (Tm) of the complete hybrid.
  • Tm melting temperature
  • bacteria inspection microarray, bacteria inspection kit, bacteria inspection probe set, and bacteria inspection primer set of the present embodiment described above the above-described inspection has not been possible so far. It is possible to specifically and accurately identify all 17 types of Listeria species including the Listeria species of (1) to (10).
  • DSM Deutsche Sammlung von Mikroorganismen
  • the PCR reaction solution is uniformly a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 1 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 2 for amplifying the IGS region of the DNA of Listeria The one containing was used.
  • These primers were synthesized by Sigma-Aldrich Sakai Japan LLC.
  • PCR buffer QIAGEN
  • dNTP mix Cat No.BR0600503, biotechrabbit
  • 10 ⁇ M forward primer SEQ ID NO: 1
  • 10 ⁇ M reverse primer SEQ ID NO: 2
  • HotStarTaq DNA Polymerase (Cat No. 203203, QIAGEN) 0.1 ⁇ l 6).
  • Template DNA (10ng / ⁇ l) 2.0 ⁇ l 7.
  • Sterile water water added until the total volume is 20.0 ⁇ l)
  • DNA was amplified under the following conditions using a nucleic acid amplification apparatus (Master cycler ep, Eppendorf).
  • A 95 ° C 15 minutes
  • b 94 ° C 1 minute
  • c 60 ° C 1 minute
  • d 72 ° C 1 minute
  • E 72 ° C 10 minutes
  • electrophoresis of amplification products by PCR was performed using an electrophoresis apparatus (MupidexU, Mupid Co., Ltd.).
  • a gel imaging device (SCOPE WD, Optima Co., Ltd.) was used as a detector for amplification products.
  • the amplification product was excised and purified.
  • HiYield Gel / PCR DNA Fragments Extraction Kit (RBC Bioscience) for excision / DNA purification was used.
  • the purified DNA was sequenced using a DNA sequence reaction apparatus (Big Dye® X X terminator, Thermo Fisher Scientific). After the sequencing, purification was performed, and electrophoresis and analysis were performed with a DNA sequencer (3130xl Genetic Analyzer, 3730xl DNA Analyzer, Thermo Fisher Scientific). These sequences, migration and analysis were performed by Fasmac Co., Ltd. As a result, the obtained base sequences are shown in FIG. 2, FIG. 3, and FIG.
  • FIG. 2 shows the nucleotide sequence in the IGS region of (1) Listeria fluoridensis, (2) Listeria wehenstephanensis, (3) Listeria cornelensis, (4) Listeria lipariae, and (5) Listeria grandensis (sequence number 3 in this order). To 7) are shown.
  • FIG. 3 shows the nucleotide sequences in the IGS region of (6) Listeria booriae, (7) Listeria nujönensis, (8) Listeria aquatica, (9) Listeria freshmanni, and (10) Listeria malti. 8-12) are shown.
  • FIG. 23 shows the base sequence (SEQ ID NO: 46) in the IGS region of (10) Listeria multi. Based on these base sequences and the like, the bacterial test probe set and bacterial test primer set of this embodiment were designed.
  • Tests for confirming the effects of the bacteria testing method, bacteria testing microarray, bacteria testing kit, bacteria testing probe set, and bacteria testing primer set of this embodiment were performed.
  • the probe set for testing bacteria according to the present embodiment was designed by comparing the IGS regions of all 17 Listeria species to identify highly specific regions. In this test, the specificity (inclusion) of these probe sets was verified.
  • Sample numbers 1 to 6 Listeria grayi ATCC 25401, ATCC 700545, ATCC 19120, ATCC 25400, ATCC 25403, ATCC 19120 ⁇ Sample Nos. 7-8: Listeria innocua in order ATCC 33090, NCTC 11288 Sample numbers 9 to 10: Listeria ivanovii subsp. Ivanovii ATCC 19119, Listeria ivanovii subsp.
  • Sample number 32 Listeria floridensis DSM 26687 Sample numbers 33 to 34: Listeria weihenstephanensis DSM 24698, DSM 24699 Sample number 35: Listeria cornellensis DSM 26689 Sample number 36: Listeria riparia DSM 26685 Sample number 37: Listeria grandensis DSM 26688 Sample number 38: Listeria booriae DSM 28860 Sample number 39: Listeria newyorkensis DSM 28861 Sample number 40: Listeria aquatica DSM 26686 Sample number 41: Listeria yakmannii subsp. Fleischmannii DSM 25003 Sample number 42: Listeria marthii DSM 23813 ⁇ Sample No. 43: Negative control (Add sterilized water instead of Template)
  • the PCR reaction solution is uniformly a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 16 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of the DNA of Listeria
  • the one containing was used.
  • an internal control was used to determine whether or not DNA amplification by PCR reaction was properly performed.
  • plasmid DNA was used, and a base sequence shown in SEQ ID NO: 15 was prepared by a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 13 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 14.
  • the target DNA consisting of was amplified.
  • These primers were synthesized by Sigma Aldrich Japan LLC.
  • the target DNA of the plasmid was synthesized by Fasmac Co., Ltd.
  • DNA was amplified under the following conditions using a nucleic acid amplification apparatus (Master cycler ep, Eppendorf).
  • A 95 ° C 15 minutes
  • b 94 ° C 1 minute
  • c 60 ° C 1 minute
  • d 72 ° C 1 minute
  • E 72 ° C 10 minutes
  • a DNA chip manufactured by Toyo Seikan Group Holdings Co., Ltd.
  • a probe with SEQ ID NOS: 18 to 45 shown in FIGS. 8 and 9 was used.
  • Each probe was synthesized by Sigma-Aldrich Sakai Japan LLC.
  • Each probe was immobilized on a substrate using a DNA spotter.
  • SEQ ID NO: 18 shows the base sequence of a probe whose detection target is Listeria.
  • SEQ ID NO: 19 shows the base sequence of a probe whose detection target is Listeria gray.
  • SEQ ID NOs: 20 to 21 show the base sequences of probes for detecting Listeria innocure.
  • SEQ ID NOs: 22 to 23 show the base sequences of probes for detecting Listeria ivanovi.
  • SEQ ID NOs: 24 to 25 show the base sequences of probes for detecting Listeria monocytogenes.
  • SEQ ID NOs: 26 to 28 show the base sequences of probes for detecting Listeria locotier.
  • SEQ ID NO: 29 shows the base sequence of a probe whose detection target is Listeria shirigeri.
  • SEQ ID NOs: 30 to 31 show the base sequences of probes for detecting Listeria well simelii.
  • SEQ ID NOs: 32 to 33 show the base sequences of probes for detecting Listeria fluoridensis.
  • SEQ ID NO: 34 is the base sequence of the probe whose detection target is Listeria admir step hanensis.
  • SEQ ID NO: 35 is the base sequence of the probe whose detection target is Listeria cornellensis.
  • SEQ ID NO: 36 shows the base sequence of a probe whose detection target is Listeria liparia.
  • SEQ ID NO: 37 shows the base sequence of a probe whose detection target is Listeria grandensis.
  • SEQ ID NOs: 38 to 39 show the base sequences of probes for detection of Listeria boriae.
  • SEQ ID NOs: 40 to 41 show the base sequences of probes for detecting Listeria New Yorkensis.
  • SEQ ID NO: 42 shows the base sequence of a probe whose detection target is Listeria aquatica.
  • SEQ ID NO: 43 shows the base sequence of a probe whose detection target is Listeria Georgiani.
  • SEQ ID NO: 44 shows the base sequence of a probe whose detection target is Listeria multi.
  • SEQ ID NO: 45 shows the base sequence of a probe whose target is a plasmid.
  • a reaction solution for hybridization was prepared as follows. 2 ⁇ L of hybridization buffer and 4 ⁇ L of amplification product were mixed on the cover of the DNA chip.
  • the buffer composition is 3 ⁇ SSC / 0.3% Tween20 and 20 nM Cy5 labeled oligonucleotide. Then, this cover was put on a DNA chip, and hybridization was performed in a hybridizer (Dako) at 45 ° C./1 hour for hybridization conditions.
  • the PCR product that had not been hybridized was washed away using a washing solution (0.5 ⁇ SSC / 0.2% SDS, 0.5 ⁇ SSC) for the DNA chip. Then, using a label detection device (GENOGATE (R) reader, Toyo Seikan Group Engineering Co., Ltd.), the fluorescence intensity of each probe placed on the DNA chip (the fluorescence intensity of the amplification product bound to the probe) is measured. Then, the S / N ratio value ((median fluorescence intensity value ⁇ background value) ⁇ background value) for each probe was obtained. The results are shown in FIGS. In these figures, when the S / N ratio value was positive for any of the probe sets corresponding to the Listeria species to be detected, it was determined that the Listeria species were detected. In these figures, positive S / N ratio values are indicated by thick frames.
  • the probe shown in SEQ ID NO: 18 is positive for all 17 Listeria species.
  • the probes shown in SEQ ID NOs: 19 to 43 are capable of detecting Listeria spp. That are the respective detection targets.
  • the probe set for testing bacteria of this embodiment exhibits excellent specificity (inclusion) except for SEQ ID NO: 44 for detecting Listeria multiplicity. Therefore, for Listeria multi, a probe shown in SEQ ID NO: 47 was newly developed. And in order to confirm the specificity (inclusion) of this embodiment including this probe, the test 4 mentioned later was done.
  • Sample number 44 Staphylococcus aureus GTC DY 0073
  • Sample number 45 Escherichia coli RIMD 0509516
  • Sample number 46 Shigella dysenteriae GTC 00100
  • Sample number 47 Nagvibrio Vibrio mimicus RIMD 2218001
  • Sample numbers 48-58 Salmonella enterica subsp salamae GTC 1731, Salmonella enterica subsp enterica serovar Arizonae GTC 1732, Salmonella enterica subsp enterica serovar Cerro GTC 2554, Salmonella enterica subsp enterica serovar Landau GTC 2614, TC 3820, Salmonalla enterica subsp enterica serovar Enteritidis GTC 3838, Salmonella enterica subsp enterica serovar Aberdeen GTC 12694, Salmonella enterica subsp enterica serovar Kentucky GTC 12786, Salmonella enterica subsp enterica serovar Essen GTC 12814, Salmonella entericaS enterica serovar Niarembe GTC 12820 ⁇ Sample No. 59: Negative control (Add sterilized water instead of Template)
  • the PCR reaction solution was composed of a forward primer consisting of the base sequence shown in SEQ ID NO: 16 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of Listeria spp.
  • the one containing the primer set was used.
  • an internal control was used to determine whether or not DNA amplification by PCR reaction was properly performed.
  • plasmid DNA was used, and a base sequence shown in SEQ ID NO: 15 was prepared by a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 13 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 14.
  • the target DNA consisting of was amplified.
  • These primers were synthesized by Sigma Aldrich Japan LLC.
  • the target DNA of the plasmid was synthesized by Fasmac Co., Ltd.
  • DNA was amplified under the following conditions using a nucleic acid amplification apparatus (Master cycler ep, Eppendorf).
  • A 95 ° C 15 minutes
  • b 94 ° C 1 minute
  • c 60 ° C 1 minute
  • d 72 ° C 1 minute
  • E 72 ° C 10 minutes
  • test microarray a DNA chip (manufactured by Toyo Seikan Group Holdings Co., Ltd.) was used as in the test 1, and probes having the sequence numbers 18 to 45 shown in FIGS. 8 and 9 were used. Each probe was synthesized by Sigma-Aldrich Sakai Japan LLC. Each probe was immobilized on a substrate using a DNA spotter.
  • a reaction solution for hybridization was prepared as follows in the same manner as in Test 1. 2 ⁇ L of hybridization buffer and 4 ⁇ L of amplification product were mixed on the cover of the DNA chip.
  • the buffer composition is 3 ⁇ SSC / 0.3% Tween20 and 20 nM Cy5 labeled oligonucleotide. Then, this cover was put on a DNA chip, and hybridization was performed in a hybridizer (Dako) at 45 ° C./1 hour for hybridization conditions.
  • the PCR product that had not been hybridized was washed away using a washing solution (0.5 ⁇ SSC / 0.2% SDS, 0.5 ⁇ SSC) for the DNA chip. Then, using a label detection device (GENOGATE (R) reader, Toyo Seikan Group Engineering Co., Ltd.), the fluorescence intensity of each probe placed on the DNA chip (the fluorescence intensity of the amplification product bound to the probe) is measured. Then, the S / N ratio value ((median fluorescence intensity value ⁇ background value) ⁇ background value) for each probe was obtained. The results are shown in FIGS. In these figures, positive S / N ratio values are indicated by thick frames.
  • the probe for internal control shown in SEQ ID NO: 45 shows positive for all the samples shown in sample numbers 44 to 59.
  • the other probes show negative for all bacteria of sample numbers 44 to 58, and no false positives are shown.
  • the probe set for testing bacteria according to this embodiment exhibits excellent specificity (exclusiveness).
  • Test 3 In this test, the specificity (simultaneous detection) of the test probe set for bacteria of this embodiment was verified using a sample in which a plurality of Listeria species were mixed.
  • Sample numbers 60 to 65 were those to which no plasmid was added as an internal control, and sample numbers 66 to 71 were samples to which an internal control was added.
  • Sample Nos. 60 and 61 Listeria Booliae, Listeria Nuyoensis, Listeria Freshmanni Sample No. 62: Listeria Floridensis, Listeria Wegenstep Hanensis, Listeria Georgiaensis, Listeria Liparia, Listeria Grandensis, Listeria Booliae, Listeria Nuyoensis , Listeria Aquatica, Listeria Freshmanni ⁇ Sample No. 63: Sample No. 62 plus Listeria Marti ⁇ Sample No. 64: Listeria genus excluding Listeria genus ⁇ Sample No. 65: All 17 types of Listeria spp. And sample numbers 66-71 contain the same strains as sample numbers 60-65, respectively. It is intended to include a roll.
  • the PCR reaction solution was composed of a forward primer consisting of the base sequence shown in SEQ ID NO: 16 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 17 for amplifying the IGS region of Listeria spp.
  • the one containing the primer set was used.
  • an internal control was used to determine whether or not DNA amplification by the PCR reaction was properly performed.
  • plasmid DNA was used, and a base sequence shown in SEQ ID NO: 15 was prepared by a primer set consisting of a forward primer consisting of the base sequence shown in SEQ ID NO: 13 and a reverse primer consisting of the base sequence shown in SEQ ID NO: 14.
  • the target DNA consisting of was amplified.
  • These primers were synthesized by Sigma Aldrich Japan LLC.
  • the target DNA of the plasmid was synthesized by Fasmac Co., Ltd.
  • PCR buffer QIAGEN
  • dNTP mix Cat No.BR0600503, biotechrabbit
  • 10 ⁇ M forward primer SEQ ID NO: 16
  • 10 ⁇ M reverse primer SEQ ID NO: 17
  • 10 ⁇ M IC forward primer SEQ ID NO: 13
  • 10 ⁇ M reverse primer for IC SEQ ID NO: 14
  • 1.0 ⁇ l 7 100 pg / ⁇ l plasmid solution (SEQ ID NO: 15) 1.0 ⁇ l 8).
  • HotStarTaq DNA Polymerase Cat No.
  • Template DNA (10ng / PCR reaction solution) 2.0 ⁇ l 10. Sterile water (water added until the total volume is 20.0 ⁇ l) Template DNA was used as a reaction solution for 10 ng / PCR of each strain in sample numbers 60 and 66, and as a reaction solution for 1 ng / PCR of each strain in sample numbers 61 to 65 and 67 to 71.
  • DNA was amplified under the following conditions using a nucleic acid amplification apparatus (Master cycler ep, Eppendorf).
  • A 95 ° C 15 minutes
  • b 94 ° C 1 minute
  • c 60 ° C 1 minute
  • d 72 ° C 1 minute
  • E 72 ° C 10 minutes
  • a DNA chip manufactured by Toyo Seikan Group Holdings Co., Ltd.
  • a probe with SEQ ID NOS: 18 to 45 shown in FIGS. did.
  • Each probe was synthesized by Sigma-Aldrich Sakai Japan LLC.
  • Each probe was immobilized on a substrate using a DNA spotter.
  • a reaction solution for hybridization was prepared as follows in the same manner as in Test 1. 2 ⁇ L of hybridization buffer and 4 ⁇ L of amplification product were mixed on the cover of the DNA chip.
  • the buffer composition is 3 ⁇ SSC / 0.3% Tween20 and 20 nM Cy5 labeled oligonucleotide. Then, this cover was put on a DNA chip, and hybridization was performed in a hybridizer (Dako) at 45 ° C./1 hour for hybridization conditions.
  • the PCR product that had not been hybridized was washed away using a washing solution (0.5 ⁇ SSC / 0.2% SDS, 0.5 ⁇ SSC) for the DNA chip. Then, using a label detection device (GENOGATE (R) reader, Toyo Seikan Group Engineering Co., Ltd.), the fluorescence intensity of each probe placed on the DNA chip (the fluorescence intensity of the amplification product bound to the probe) is measured. Then, the S / N ratio value ((median fluorescence intensity value ⁇ background value) ⁇ background value) for each probe was obtained. The results are shown in FIGS. In these figures, when the S / N ratio value was positive for any of the probe sets corresponding to the Listeria species to be detected, it was determined that the Listeria species were detected. In these figures, positive S / N ratio values are indicated by thick frames.
  • Test 4 Including a new probe shown in SEQ ID NO: 47 for detecting Listeria multi, a method for testing bacteria according to the present embodiment, a microarray for testing bacteria, a kit for testing bacteria, a probe set for testing bacteria, and A test for confirming the specificity (inclusion) of the test primer set was conducted in the same manner as in Test 1.
  • the same samples as the sample numbers 1 to 43 in the test 1 were prepared as the sample numbers 1a to 43a, respectively, and cultured in the same manner as the test 1. Then, PCR was performed in the same manner as in Test 1 using the genomic DNA extracted from the Listeria spp.
  • the probe shown in SEQ ID NO: 18 is positive for all 17 Listeria species.
  • the probes shown in SEQ ID NOs: 19 to 43 and 47 are capable of detecting Listeria spp. That are the detection targets.
  • the probe set for testing bacteria according to the present embodiment exhibits excellent specificity (inclusion).
  • Test 5 Including the probe shown in SEQ ID NO: 47 for detecting Listeria multi, the method for testing bacteria of this embodiment, the microarray for testing bacteria, the kit for testing bacteria, the probe set for testing bacteria, and the A test for verifying the specificity (exclusiveness) of the test primer set was conducted in the same manner as in Test 2.
  • the same samples as the sample numbers 44 to 59 in the test 2 were prepared as the sample numbers 44a to 59a, respectively, and cultured in the same manner as the test 2. Then, PCR was performed in the same manner as in Test 2 using the genomic DNA extracted from Listeria spp.
  • the probe for internal control shown in SEQ ID NO: 45 is positive for all the samples shown in sample numbers 44a to 59a.
  • the other probes are negative for all the bacteria of sample numbers 44a to 58a and no false positives are shown.
  • the probe set for testing bacteria according to this embodiment exhibits excellent specificity (exclusiveness).
  • Test 6 Including a new probe shown in SEQ ID NO: 47 for detecting Listeria multi, a method for testing bacteria according to the present embodiment, a microarray for testing bacteria, a kit for testing bacteria, a probe set for testing bacteria, and A test for verifying the specificity (simultaneous detection) by the test primer set was conducted in the same manner as in Test 3.
  • sample numbers 63, 65, 69, and 71 in the test 3 are prepared as the sample numbers 63a, 65a, 69a, and 71a, respectively. And cultured in the same manner.
  • Sample numbers 72a and 73a were prepared as negative controls (sterilized water was added instead of Template).
  • Sample number 72a is a sample to which no plasmid was added as an internal control
  • sample number 73a was a sample to which an internal control was added.
  • PCR was performed in the same manner as in Test 3 using genomic DNA extracted from the Listeria spp.
  • the present invention is not limited to the above-described embodiments and examples, and it goes without saying that various modifications can be made within the scope of the present invention.
  • the probes arranged in the microarray for testing bacteria of this embodiment are not limited to one spot per type, and a plurality of each probe may be arranged.
  • other probes for detecting bacteria other than Listeria belonging to the detection target of the present embodiment may be arranged in the microarray together with the probes in the present embodiment, and can be changed as appropriate. .
  • the present invention can be suitably used for specific and multiplex detection of Listeria spp. In environmental inspections, food inspections, and the like.

Landscapes

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

Abstract

Dans un examen pour déterminer la présence d'une bactérie, une bactérie existante appartenant au genre Listeria peut être détectée de manière très précise et spécifique. L'invention concerne un procédé d'examen de la présence d'une bactérie dans un échantillon, la bactérie à détecter étant sélectionnée parmi des bactéries (1) à (10) appartenant au genre Listeria, c'est-à-dire (1) Listeria floridensis, (2) Listeria weihenstephanensis, (3) Listeria cornellensis, (4) Listeria riparia, (5) Listeria grandensis, (6) Listeria booriae, (7) Listeria newyorkensis, (8) Listeria aquatica, (9) Listeria fleischmannii et (10) Listeria marthii, ledit procédé comprenant : (a) une étape de préparation pour préparer un mélange réactionnel liquide contenant un ensemble d'amorces pour amplifier la région d'espaceur intergénique (IGS) du gène d'ARNr ; et (b) une étape d'amplification pour amplifier l'acide nucléique de la bactérie à détecter qui est contenu dans l'échantillon à l'aide du mélange réactionnel liquide obtenu dans l'étape de préparation.
PCT/JP2019/019446 2018-05-18 2019-05-16 Procédé d'examen d'une bactérie, micropuce pour l'examen d'une bactérie, kit d'examen de bactérie, ensemble des sondes pour l'examen d'une bactérie et ensemble d'amorces pour l'examen d'une bactérie WO2019221219A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2018-096638 2018-05-18
JP2018096638 2018-05-18
JP2018190417A JP2019201626A (ja) 2018-05-18 2018-10-05 細菌の検査方法、細菌の検査用マイクロアレイ、細菌の検査用キット、細菌の検査用プローブセット、及び細菌の検査用プライマーセット
JP2018-190417 2018-10-05

Publications (1)

Publication Number Publication Date
WO2019221219A1 true WO2019221219A1 (fr) 2019-11-21

Family

ID=68539747

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/019446 WO2019221219A1 (fr) 2018-05-18 2019-05-16 Procédé d'examen d'une bactérie, micropuce pour l'examen d'une bactérie, kit d'examen de bactérie, ensemble des sondes pour l'examen d'une bactérie et ensemble d'amorces pour l'examen d'une bactérie

Country Status (1)

Country Link
WO (1) WO2019221219A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961926A (zh) * 2021-03-18 2021-06-15 广东省科学院微生物研究所(广东省微生物分析检测中心) 一种同时检测单增李斯特菌cc87和88型菌株的引物、试剂盒以及方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRAHAM, TA. ET AL.: "Inter-and Intraspecies Comparison of the 16S-23S rRNA Operon Intergenic Spacer Regions of Six Listeria spp.", INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, vol. 47, no. 3, 1997, pages 863 - 869, XP055654389 *
LIN, MC . ET AL.: "Use of oligonucleotide array for identification of six foodborne pathogens and Pseudomonas aeruginosa grown on selective media", J FOOD PROT, vol. 68, no. 11, 2005, pages 2278 - 86, XP009103178 *
O'CONNOR L. ET AL.: "Rapid polymerase chain reaction/DNA probe membrane-based assay for the detection of Listeria and Listeria monocytogenes in food", J FOOD PROT, vol. 63, no. 3, 2000, pages 337 - 42, XP009009531 *
ORSI, RH. ET AL.: "Characteristics and distribution of Listeria spp., including Listeria species newly described since 2009", APPL MICROBIOL BIOTECHNOL, vol. 100, no. 12, 2016, pages 5273 - 87, XP035870496 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112961926A (zh) * 2021-03-18 2021-06-15 广东省科学院微生物研究所(广东省微生物分析检测中心) 一种同时检测单增李斯特菌cc87和88型菌株的引物、试剂盒以及方法
CN112961926B (zh) * 2021-03-18 2024-01-09 广东省科学院微生物研究所(广东省微生物分析检测中心) 一种同时检测单增李斯特菌cc87和88型菌株的引物、试剂盒以及方法

Similar Documents

Publication Publication Date Title
JP3097059B2 (ja) 標的ヌクレオチド配列に対する特異的プローブの生成
Kim et al. Microarray detection of food-borne pathogens using specific probes prepared by comparative genomics
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
Kim et al. Detection of representative enteropathogenic bacteria, Vibrio spp., pathogenic Escherichia coli, Salmonella spp., Shigella spp., and Yersinia enterocolitica, using a virulence factor gene-based oligonucleotide microarray
JP2024069451A (ja) サルモネラ属菌血清型判別用プライマーセット、ｐｃｒ産物、サルモネラ属菌血清型判別用マイクロアレイ、サルモネラ属菌血清型判別用キット、及びサルモネラ属菌血清型判別方法
EP4130272A1 (fr) Ensemble d'amorces et sonde pour détection de bactéries du genre klebsiella
Hu et al. Simultaneous analysis of foodborne pathogenic bacteria by an oligonucleotide microarray assay
Debruyne et al. Comparative performance of different PCR assays for the identification of Campylobacter jejuni and Campylobacter coli
WO2019221219A1 (fr) Procédé d'examen d'une bactérie, micropuce pour l'examen d'une bactérie, kit d'examen de bactérie, ensemble des sondes pour l'examen d'une bactérie et ensemble d'amorces pour l'examen d'une bactérie
US20100167956A1 (en) Dna chip for detection of escherichia coli
JP2017136019A (ja) 病原性大腸菌検出用担体、病原性大腸菌検出用キット、及び病原性大腸菌の検出方法
JP5048622B2 (ja) 乳酸菌検出用ｐｃｒプライマー
US20170081709A1 (en) Method for detecting escherichia coli and carrier for detecting escherichia coli
Saint et al. A PCR test for the identification and discrimination of Legionella longbeachae serogroups 1 and 2
KR102407237B1 (ko) 캠필로박터를 선택적으로 구분 검출할 수 있는 리얼-타임 pcr 키트
KR101299627B1 (ko) 식중독 세균 동시 검출용 프라이머 및 이의 용도
JP6648559B2 (ja) 細菌の検査方法、プライマーセット、細菌検査用担体、及び細菌検査用キット
JP2019201626A (ja) 細菌の検査方法、細菌の検査用マイクロアレイ、細菌の検査用キット、細菌の検査用プローブセット、及び細菌の検査用プライマーセット
AU776138B2 (en) Detection of mycobacterium avium subspecies
WO2016129249A1 (fr) Procédé pour détecter des bactéries d'acide lactique, kit de détection de bactéries d'acide lactique et instrument de détection de bactéries d'acide lactique
US20100167951A1 (en) Dna chip for detection of staphylococcus aureus
EP2723891B1 (fr) Méthodes de diagnostic pour la détection de clostridium difficile
KR20230095302A (ko) 조류 클라미디아균 감별 동정용 실시간 유전자 진단법
JP6672760B2 (ja) 病原性大腸菌検出用担体、病原性大腸菌検出用キット、及び病原性大腸菌の検出方法
JP3331977B2 (ja) 赤痢菌検出のためのオリゴヌクレオチドおよびそれを用いた検出法

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: 19803296

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: 19803296

Country of ref document: EP

Kind code of ref document: A1