CN106434898A - Method, primers and kit for quickly detecting yersinia pseudotuberculosis at constant temperature - Google Patents

Method, primers and kit for quickly detecting yersinia pseudotuberculosis at constant temperature Download PDF

Info

Publication number
CN106434898A
CN106434898A CN201610780457.8A CN201610780457A CN106434898A CN 106434898 A CN106434898 A CN 106434898A CN 201610780457 A CN201610780457 A CN 201610780457A CN 106434898 A CN106434898 A CN 106434898A
Authority
CN
China
Prior art keywords
primer
sequence
primer sets
sets
yersinia genus
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
CN201610780457.8A
Other languages
Chinese (zh)
Other versions
CN106434898B (en
Inventor
李园园
李雪玲
韦朝春
贾犇
刘伟
陆长德
李亦学
曹永梅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Wang Wang Food Group Co Ltd
Shanghai Industrial Institute For Research And Technology
Original Assignee
Shanghai Wang Wang Food Group Co Ltd
Shanghai Industrial Institute For Research And Technology
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 Shanghai Wang Wang Food Group Co Ltd, Shanghai Industrial Institute For Research And Technology filed Critical Shanghai Wang Wang Food Group Co Ltd
Priority to CN202010036128.9A priority Critical patent/CN111020046A/en
Priority to CN202010035792.1A priority patent/CN111100906A/en
Publication of CN106434898A publication Critical patent/CN106434898A/en
Application granted granted Critical
Publication of CN106434898B publication Critical patent/CN106434898B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

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
    • C12Q1/6844—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical 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)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a method, primer group and kit for quickly detecting yersinia pseudotuberculosis at constant temperature. The method comprises the steps that genomic DNA is extracted from a sample to be detected; a constant-temperature amplification reaction is conducted by taking the genomic DNA as a template and taking the primer group capable of amplifying the specific sequences of the yersinia pseudotuberculosis as primers in an enzyme reaction system; whether the yersinia pseudotuberculosis exists in the sample to be detected or not is determined by judging whether a reaction result is positive or not. The detection method has the advantages that the high sensitivity and specificity are achieved, the detection time is short, result judging is easy, operation is convenient and rapid, the cost is low, and a wide application prospect is achieved.

Description

The method of fast constant temperature detection artificial tuberculosis yersinia genus, primer and kit
Technical field
The invention belongs to biological technical field, be specifically related to the side of a kind of fast constant temperature detection artificial tuberculosis yersinia genus Method, primer and kit.
Background technology
Artificial tuberculosis yersinia genus (Yersinia pseudotuberculosis) belongs to enterobacteriaceae Yersinia ruckeri Belonging to, being the enteric pathogenic bacteria of a kind of infecting both domestic animals and human, this bacterium can survive at low ambient temperatures, and therefore refrigerator storage food is modern society The important infection sources that this bacterium infects can occur, gastrointestinal symptom, mesenteric lymphadenitis etc. can be caused.This bacterium infects people In Qun to distribute based on, also can cause breaking out of different scales once in a while.Due to its atypical symptoms, easily cause diagnosis not Bright even mistaken diagnosis and delay treatment.Therefore, for the prevention of this bacterium with detect extremely important.
Tradition artificial tuberculosis yersinia genus detection method is longer due to the detection cycle, operates relative complex, and detection efficiency is relatively Low, it is difficult to meet modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, convenient Requirement.Recently as the development of nucleic acid molecules detection technique, researcher have also been developed the detection means such as PCR, but The method needs special detecting instrument, therefore, is not appropriate for being widely used in detection department of basic unit especially enterprise's production line The real-time on-site detection that inside is carried out.In order to ensure food security, be badly in need of quick, simple, accurately method detect in food Artificial tuberculosis yersinia genus.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is in recent years A kind of novel constant-temperature nucleic acid amplification method growing up, this method is for 4 specific primers of 6 region designs of target sequence (including upstream and downstream outer primer F3 and B3 and upstream and downstream inner primer FIP and BIP, wherein FIP is made up of F1C and F2, and BIP is by B1C With B2 composition), utilize a kind of archaeal dna polymerase with strand-displacement activity, be incubated about 60min at constant temperature, core can be completed Acid amplified reaction, produces macroscopic byproduct of reaction-white magnesium pyrophosphate and precipitates (see document Notomi T, Okayama H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.Loop-mediated isothermal amplification of DNA,Nucleic Acids Research,2000Jun 15；28(12):E63).This technology has Not needing PCR instrument or quantitative real time PCR Instrument, can completing under constant temperature, naked eyes i.e. can determine whether reaction result, and highly sensitive, High specificity, reaction time is short, simple operation, low cost and other advantages.
Design of primers is a step the most key in LAMP technology, and Normal practice is by the spy generally acknowledging of certain biology to be detected Specific gene imports the online website (http of LAMP primer design://primerexplorer.jp/e), set relevant parameter raw Become primer sets.It is to say, user is it is first necessary to guarantee the distinguished sequence that this target gene is species to be measured.With patent of invention CN As a example by 101182575B and CN 101200760A, they are respectively directed to the special sequence of the artificial tuberculosis yersinia genus of document report District and gyrB gene between row 16S-23S, use LAMP technology to carry out artificial tuberculosis yersinia genus detection.But, so-called " public The specific gene recognized " is often based upon delayed knowledge, and is not based on ever-increasing microbial genome data and carries out necessity Renewal, cause the primer obtaining based on this target-gene sequence not necessarily to can ensure that it is specific and/or logical in actual applications The property used.Present invention table 1 illustrate primer specificity present in prior art not, the problem that can not guarantee of versatility. It is to say, the artificial tuberculosis yersinia genus detection sequence actually not pseudoconcretion yersinia genus used in art methods Salmonella institute is peculiar, i.e. be possible to non-artificial tuberculosis yersinia genus is regarded as artificial tuberculosis yersinia genus mistakenly.Similar asks Topic exists in the confirmation of versatility, i.e. be possible to the part bacterial strain of missing inspection artificial tuberculosis yersinia genus.Therefore, in industry urgently A kind of need to be able to ensure that specific and versatility artificial tuberculosis yersinia genus detection method, meet detection department pair of basic unit simultaneously Quickly, demand easily, can carry out real-time on-site detection easily inside enterprise's production line.
Content of the invention
The technical problem to be solved in the present invention is to overcome primer versatility present in existing LAMP technology design of primers With specific not enough defect, make full use of microbial genome sequence information abundant in current common data resource and phase The sequence analysis tools answered, is designed for the primer sets of specific recognition artificial tuberculosis yersinia genus, and is formed on this basis High sensitivity, high specific detection kit.The present invention is based on the microbial genome data resource in GenBank database (by data on August 5th, 2013) carry out the design of artificial tuberculosis yersinia genus LAMP primer, provide a kind of fast constant temperature and expand Increase method, primer sets and the kit of detection artificial tuberculosis yersinia genus.Use the detection method detection pseudoconcretion of the present invention Ademilson Salmonella, has high sensitivity and a high specific, and the detection time is short, and result judges simple, simple operation, low cost excellent Point.
The present invention proposes a kind of method of quick detection artificial tuberculosis yersinia genus bacterial strain, and described method includes following step Suddenly：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that artificial tuberculosis yersinia genus genome specificity base sequence can be expanded Primer sets be primer, under enzyme reaction system, carry out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine in testing sample whether there is artificial tuberculosis yersinia genus.
The method of Constant Temperature Detection artificial tuberculosis yersinia genus bacterial strain of the present invention, extracts genomic DNA from testing sample, with Which is template, with artificial tuberculosis yersinia genus specificity amplification primer group as primer, carry out isothermal amplification reactions, then, pass through Judge whether reaction result is positive, determine in testing sample whether there is artificial tuberculosis yersinia genus.Wherein, described enzyme reaction System includes but is not limited to DNA polymerase reaction system.
In the present invention, it is 153946813 that described artificial tuberculosis yersinia genus genome specificity base sequence is No. GI 768567～768785bp bit sequence of artificial tuberculosis yersinia genus.
In the present invention, the described primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence is described A part for the nucleotide sequence of 768567～768785bp position of genome (No. GI is 153946813) or one of its complementary strand Point.Wherein, described artificial tuberculosis yersinia genus genome specificity base sequence refers to only artificial tuberculosis yersinia genus gene Group is specific, and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand artificial tuberculosis yersinia genus genome specific base sequence includes but is not limited to Primer sets A, or selected from being 50% and above primer with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence Any one group of group.
Primer sets A：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’
(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’
(SEQ ID NO：4).
In the present invention, the described primer sets that can expand artificial tuberculosis yersinia genus genome specific base sequence can also be wrapped Including with wall scroll sequence homology in aforementioned each primer sets sequence or its complementary strand sequence is 50% and above primer sets, this primer Group includes but is not limited to following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’(SEQ ID NO：5) (with primers F 3_A 5 '- GTTGTTGTATGAATTGCTGG-3 ' homology is 50%)；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’
(SEQ ID NO：7)；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’
(SEQ ID NO：8).
In the inventive method, the described primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence can To comprise or not comprise ring primer.Described ring primer can be one or more, including primer LF and/or LB.Described energy amplification The primer sets of artificial tuberculosis yersinia genus genome specificity base sequence is selected from following primer sets A ', any one group of B '；Or Selected from and described primer sets A ', in B ' sequence or its complementary strand sequence wall scroll sequence homology be 50% and above primer sets it Any one group：
Primer sets A '：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_A：5’-TATGCTCATCCAGGCGTTTC-3’(SEQ ID NO：9)；
And/or, lower lantern primer LB_A：5’-TGATGGCGATGGGGAAAATT-3’(SEQ ID NO：10)；
Primer sets B '：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_B：5’-GGGATTTCAATATGCTCATCCAG-3’(SEQ ID NO：11)；
And/or, lower lantern primer LB_B：5’-TGATGGCGATGGGGAAAATT-3’(SEQ ID NO：12).
In a particular embodiment, for example, above-mentioned primer sets A ', in B ', can only comprise a upper lantern primer, or only Comprise a lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.
In the inventive method, in a specific embodiments (primer containing ring), the enzyme reaction system of described constant-temperature amplification is： 1 × BstDNA polymeric enzyme reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L dNTP, 0.8- FIP and the BIP primer of 2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, LF and/or LB of 0.4-1.0 μm of ol/L draws Thing, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.(draw without ring in another specific embodiments Thing) in, the enzyme reaction system of described constant-temperature amplification is：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+ (MgSO4Or MgCl2), FIP and the BIP primer of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L, 0.15-0.3 μm of ol/L's F3 and B3 primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.Ring primer is favorably improved reaction Efficiency.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, comprises 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,2mM MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With the magnesium ion Mg in enzyme reaction system2+Do merging treatment.
In the inventive method, the response procedures of described isothermal amplification reactions hatches 10～90min, preferably for 1. 60～65 DEG C Ground is 10～60min；2. 80 DEG C terminate reaction 2～20min.The present invention does not limit and realizes this by other suitable response procedures Invention detection method.
In the inventive method, detection method includes but is not limited to electrophoresis detection, Turbidity measurement or color developing detection etc..Described electricity Swimming detection, preferably gel electrophoresis assays, can be Ago-Gel, it is also possible to be polyacrylamide gel.Electrophoresis detection In result, band as stepped in electrophoretogram expression characteristics, then testing sample is that artificial tuberculosis yersinia genus is positive, containing false knot Core Yersinia ruckeri；Band as stepped in electrophoretogram not expression characteristics, then testing sample is that artificial tuberculosis yersinia genus is negative. Described Turbidity measurement, is to detect by an unaided eye or transmissometer detection turbidity, and detection pipe occurs substantially muddy, then testing sample is false knot Core Yersinia ruckeri is positive, containing artificial tuberculosis yersinia genus；As muddy in having no, then testing sample is artificial tuberculosis yersinia genus Negative.Also can visually observe whether have precipitation at the bottom of reaction tube after centrifugal, if there being precipitation at the bottom of reaction tube, then testing sample is vacation Tuberculosis Yersinia ruckeri is positive, containing artificial tuberculosis yersinia genus；Do not precipitate as at the bottom of reaction tube, then testing sample is pseudoconcretion Yersinia ruckeri is negative.
Described color developing detection, is addition developer, including but not limited to calcein (50 μM) or SYBR in reaction tube GreenI (30-50 ×), or hydroxynaphthol blue (i.e. HNB, 120-150 μM).Make when using calcein or SYBR Green I During for developer, as after reaction, color is orange, then testing sample is that artificial tuberculosis yersinia genus is negative；As after reaction, color is Green, then testing sample is that artificial tuberculosis yersinia genus is positive, containing artificial tuberculosis yersinia genus.Make when using hydroxynaphthol blue During for developer, as after reaction, color is pansy, then testing sample is that artificial tuberculosis yersinia genus is negative；Such as face after reaction Look is sky blue, then testing sample is that artificial tuberculosis yersinia genus is positive.Described color developing detection, except above by visually observing Outside reaction result, it is also possible to carry out real-time or end point determination reaction result by detecting instrument, set feminine gender instead by rational The threshold value answered, when the result of testing sample reaction is less than or equal to this threshold value, then testing sample is artificial tuberculosis yersinia genus Negative；When the result of testing sample reaction is more than this threshold value, then testing sample is that artificial tuberculosis yersinia genus is positive.Described inspection Survey instrument include but is not limited to sepectrophotofluorometer, quantitative real time PCR Instrument, constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument and Genie II isothermal duplication fluorescence detecting system etc..
In described color developing detection, according to calcein or hydroxynaphthol blue as developer, can be anti-at constant-temperature amplification Added before should, it is also possible to add after isothermal amplification reactions completes, it is therefore preferable to add before isothermal amplification reactions, permissible The effective possibility reducing reaction pollution.According to SYBR Green I as developer, then complete in isothermal amplification reactions Add afterwards.According to calcein as developer, then while adding 50 μM of calceins in enzyme reaction system, add 0.6-1mM[Mn2+], for example, the MnCl of 0.6-1mM2.
Present invention also offers for the primer in the method for Constant Temperature Detection artificial tuberculosis yersinia genus bacterial strain.Described primer Including the primer sets of artificial tuberculosis yersinia genus genome specific base sequence can be expanded, it includes but is not limited to, described primer Sequence be the nucleotide sequence of 768567～768785bp position of the artificial tuberculosis yersinia genus genome that No. GI is 153946813 A part or the part of its complementary strand.
Wherein, the described primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence is selected from following Any one group of primer sets, or selected from being 50% with wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence And above arbitrary primer sets.Wherein, described primer sets includes but is not limited to following primer sets A.Described with aforementioned primer sets sequence In row or its complementary strand sequence, wall scroll sequence homology is 50% and above primer sets includes but is not limited to following primer sets B.
Primer sets A：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’.
Primer sets B：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’.
The present invention in the primer in described Constant Temperature Detection artificial tuberculosis yersinia genus method, described can expand pseudoconcretion The primer sets of Yersinia ruckeri genome specificity base sequence can also comprise or not comprise one or more ring primer；Described Ring primer is LF and/or LB.The described primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence is selected from Following primer sets A ', any one group of B '；Or be selected from and described primer sets A ', wall scroll sequence in B ' sequence or its complementary strand sequence Homology is 50% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_A：5’-TATGCTCATCCAGGCGTTTC-3’；
And/or, lower lantern primer LB_A：5’-TGATGGCGATGGGGAAAATT-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_B：5’-GGGATTTCAATATGCTCATCCAG-3’；
And/or, lower lantern primer LB_B：5’-TGATGGCGATGGGGAAAATT-3’.
In a particular embodiment, above-mentioned primer sets A ', in B ', can only comprise a upper lantern primer, or only comprise Article one, lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.In one embodiment, institute State primer and be respectively the primer shown in FIP, BIP, F3, B3, LF and LB or single with aforementioned primer sequence or its complementary strand sequence Bar primer homology is 50% and above primer.
The present invention also provides a kind of for the kit in above-mentioned Constant Temperature Detection artificial tuberculosis yersinia genus bacterial strain method, its Including the described primer sets that can expand artificial tuberculosis yersinia genus genome specific base sequence.In kit of the present invention, described The primer sets of artificial tuberculosis yersinia genus genome specificity base sequence can be expanded, including but not limited to genome (No. GI: 153946813) part for nucleotide sequence for 768567～768785bp position or a part for its complementary strand are drawn as described Thing sequence；Described primer includes but is not limited to described primer sets A.Also including but not limited to with aforementioned primer sequence or it is complementary In chain-ordering, wall scroll sequence homology is 50% and above primer sets is as primer；Including but not limited to primer sets B.
In kit of the present invention, the described primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence Can comprise or not comprise one or more ring primer；Ring primer is as optional component.Described ring primer is LF and/or LB.Bag The primer sets of the LF and/or LB of primer containing ring includes but is not limited to primer sets A ', B ' etc..In a particular embodiment, present invention examination Agent box can comprise LF and/or the LB ring primer of 0.4-1.0 μm of ol/L.In one embodiment, the sequence of primer sets Be respectively the primer shown in FIP, BIP, F3, B3, LF and LB or with foregoing sequences or its complementary strand sequence wall scroll primer homology It is 50% and above primer.
In kit of the present invention, also include Bst DNA polymerase buffer liquid, Bst archaeal dna polymerase, dNTP solution, Mg2+ (MgSO4Or MgCl2) and glycine betaine in one or more.In one embodiment, kit enzyme reaction system of the present invention Comprise 1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/LdNTP, FIP and the BIP primer of 0.8-2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst DNA Polymerase and the glycine betaine of 0-1.5mol/L.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, comprises 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%TritonX-100,2mM MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With enzyme reaction body Magnesium ion Mg in system2+Do merging treatment.
In kit of the present invention, also comprise positive control template.In one embodiment, described positive control template Include but is not limited to complete genome DNA, portion gene group DNA of artificial tuberculosis yersinia genus, or comprise pseudoconcretion pestis Bacterium complete genome DNA or the carrier of portion gene group DNA.
In kit of the present invention, also comprising negative control template, described negative control template includes but is not limited to distilled water.
In kit of the present invention, also comprising developer, developer includes but is not limited to calcein, SYBR Green I or Hydroxynaphthol blue.When developer is calcein, kit also comprises [Mn2+], for example, MnCl2.
In kit of the present invention, also comprise distilled water.
In kit of the present invention, also comprise nucleic acid extraction reagent.
The invention allows for a kind of carrier, described carrier comprises selected from primer sets A, B, A ', any one group of primer of B '. This carrier has the specific DNA sequence dna of artificial tuberculosis yersinia genus owing to containing, therefore can be applicable to microbial taxonomy, The research field such as comparative genomics, evolution, and the application such as microorganism detection.This carrier can be but not limited to plasmid Carrier (such as pBR322, pUC18, pUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda etc.) and people Make chromosome vector (such as Bacterial artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, primer sets A is comprised arbitrarily Article one, carrier pBR322-A, the carrier pBR322-B ... of any one primer comprising primer sets B of primer comprises primer sets The carrier pBR322-B ' of any one the primer of B '.Carrier bacteriophage lambda-the A of any one the primer comprising primer sets A, comprise Carrier bacteriophage lambda-the B ... of any one primer of primer sets B comprise primer sets B ' the carrier λ of any one primer bite Thalline-B ' etc..
The invention allows for selected from primer sets A, B, A ', the primer of any one group of B ' is at Constant Temperature Detection pseudoconcretion Yale Application in gloomy Salmonella.
The invention allows for application in Constant Temperature Detection artificial tuberculosis yersinia genus for the described kit.
The invention allows for application in Constant Temperature Detection artificial tuberculosis yersinia genus for the described carrier.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection pseudoconcretion yersinia genus The method of Salmonella, primer/primer sets, detection reagent/kit, have greater significance to the food security of China.The present invention has Benefit effect includes：Artificial tuberculosis yersinia genus detection method of the present invention is used to have high specificity, highly sensitive, detection time Short, result judges simple, simple operation, low cost and other advantages.Compared with conventional at present detection method, the constant temperature that the present invention uses TRAP, can be carried out under constant temperature, only need to use simple thermostat, it is not necessary to the expensive instrument in PCR experiment, Do not need to carry out amplified production the steps such as electrophoresis detection, thus, it is very suitable for being widely used in various circles of society and include that basic unit eats Product safety detection department promotes the use of, even if also may be used in the environment of molecular biology professional knowledge and skills base relative deficiency Fully application.Above-mentioned each optimum condition can be combined based on common sense in the field, all belong to scope.
Brief description
Fig. 1 shows the specific of the embodiment of the present invention 7 artificial tuberculosis yersinia genus Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 artificial tuberculosis yersinia genus detection method.
Detailed description of the invention
Being combined to lower specific embodiments and the drawings, the present invention is described in further detail, the protection content of the present invention It is not limited to following example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change Change and advantage is all included in the present invention, and with appending claims as protection domain.Implement the present invention process, Condition, reagent, experimental technique etc., outside the lower content mentioned specially, be universal knowledege and the common knowledge of this area, The present invention is not particularly limited content.
Embodiment 1-6 artificial tuberculosis yersinia genus isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Derive from Chinese medicine bacteria culture preservation administrative center, numbering for the artificial tuberculosis yersinia genus bacterial classification of detection CMCC53504.Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract kit extraction gene Group DNA, DNA OD260/OD280Being 1.8, concentration is 18.94ng/ μ L.
(2) with artificial tuberculosis yersinia genus genomic DNA to be measured as template, be respectively adopted autogamy kit (be shown in Table 2, Table 3), and according to condition described in table 3, prepare reaction system, with artificial tuberculosis yersinia genus specificity amplification primer group for drawing Thing, carries out isothermal amplification reactions.Primer in embodiment 1～6 is respectively primer sets A, and A ' (1 ring primer), (2 ring draws A ' Thing), B, B ' (2 ring primer), B ' (1 ring primer).
(3) according to condition described in table 3, by electrophoresis detection, Turbidity measurement or color developing detection, amplification is carried out true Recognize.
As can be seen from Table 3, detection method and the primer sets being used and reaction system thereof can be right well Artificial tuberculosis yersinia genus specific fragment carries out expanding and obtains testing result.Additionally, when using detector to detect, Shorten the reaction time to also there being good Detection results (such as embodiment 6) during 10min.Therefore, present invention could apply to detection Whether sample contains artificial tuberculosis yersinia genus.
Embodiment 7 artificial tuberculosis yersinia genus specific detection
Collect non-artificial tuberculosis yersinia genus 29 strain (in table 4 and Fig. 1 1～24,26～30), by these bacterial strains and false knot Core pestis strain (in table 4 and Fig. 1 25) is cultivated respectively, takes 1mL bacterium solution, uses kit IA, extracts bacterium DNA, and with reference to the reaction system of embodiment 1 and condition, carry out LAMP amplification (primer sets is A) respectively and add developer sight Examine.
Its testing result is as shown in table 4 and Fig. 1, and in Fig. 1,1-24 is respectively staphylococcus aureus, Staphylococcus aureus The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, bacillus cereus, gill fungus sample bacillus, listeria monocytogenes, Ying Nuoke Listeria, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, B-mode Salmonella paratyphi, shigella dysenteriae, Shigella bogdii, shigella flexneri, ETEC (contain clostridium botulinum A type gene), pathogenic ETEC, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic big Intestines Escherichia, hemorrhagic ETEC, the rugged Cronobacter sakazakii of slope and yersinia enterocolitica, 26-30 is respectively For Vibrio vulnificus, vibrio parahaemolytious, Freund vibrios, comma bacillus and Song Shi Shigella, NTC：Negative control, 25：Pseudoconcretion Yersinia ruckeri.In Fig. 1, only the product after artificial tuberculosis yersinia genus bacterial strain amplified reaction is rendered as bright green, for the positive As a result, as shown in No. 25 pipe.And the product after other non-artificial tuberculosis yersinia genus bacterial strains and negative control amplified reaction is equal It is rendered as orange, is negative findings, as the 1st～No. 24,26～No. 30 is managed and shown in NTC negative control pipe.
By Fig. 1 and Biao 4 result it can be seen that detection kit of the present invention and detection method have good pseudoconcretion Yale Gloomy Salmonella strain specificity, i.e. the only artificial tuberculosis yersinia genus bacterial strain amplification positive, other non-artificial tuberculosis yersinia genus bacterium Strain is feminine gender.
Preparation detection kit, the primer using in kit is respectively primer sets B, primer sets A ', B ', by above-mentioned special Property detection method, respectively obtains same testing result, i.e. non-artificial tuberculosis yersinia genus bacterial strain and negative control amplified reaction After product be negative findings, the product after artificial tuberculosis yersinia genus bacterial strain amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively to primer sets A, primer sets B, primer sets A ', B ' is specifically carried out Theory analysis, it was found that in the case that each bar primer at most allows three mispairing, each primer sets has five and draws at most simultaneously Thing comparison, on non-artificial tuberculosis yersinia genus, shows the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
As described in Example 1 extract bacterium CMCC 53504 DNA, use kit IB, and according to 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg DNA ladder degree adds reaction system, and other reaction conditions are with reference to the side of table 3 embodiment 1 Method carries out LAMP amplification (primer sets is A) respectively and adds developer to observe.As in figure 2 it is shown, 1-7 be respectively 50ng, 5ng, 500pg, 50pg, 5pg, 500fg and 50fg, ntc：Negative control.The product that in Fig. 2,50ng, 5ng, 500pg are processed presents For bright green, being positive findings, 50pg, 5pg, 500fg, 50fg are processed and the product of negative control is rendered as orange, are Negative findings.Testing result shows, minimum in each reaction tube (is approximately equivalent to 10 containing 500pg5Individual bacterium) DNA when still can quilt Detection.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, primer sets A respectively ', B ', each reaction In pipe, the DNA of as little as 5pg～500fg still can be detected.
Embodiment 9 versatility detects
According to method described in table 1, respectively to primer sets A, primer sets B, primer sets A ', the versatility of B ' carries out theoretical point Analysis, it was found that the primer region of each primer sets and 4 strain artificial tuberculosis yersinia genus (No. GI is respectively 51594359, 153946813,170022262 and 186893344) coupling completely, may be used for above-mentioned 4 strain pseudoconcretion pestis in theory The detection of bacteria strain, shows that the versatility of each primer sets is all preferable.
The versatility of primer and specifically analysis in the existing detection method of table 1 artificial tuberculosis yersinia genus
Note：A) 4 genome (GI difference by the sequence between primers F in patent 3 and B3 and artificial tuberculosis yersinia genus It is 153946813,51594359,170022262 and 186893344) carry out Bowtie comparison, determine detection region at No. GI Position in 153946813 genomes.B) detection regional sequence is carried out in common data base resource Blast comparison, primer It is good that region is mated completely for versatility.C) detection regional sequence is carried out in common data base resource Blast comparison, guiding region Territory matching degree is higher, specifically poorer；If primer can not simultaneously comparison in non-artificial tuberculosis yersinia genus strain, show special Property is good.
The kit species of table 2 Constant Temperature Detection artificial tuberculosis yersinia genus and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention artificial tuberculosis yersinia genus and detection knot Really
Table 4 tests bacterial strain uses therefor and testing result
Note：a)CGMCC：China General Microbiological DSMZ, CICC：Chinese industrial Microbiological Culture Collection manages Center, CMCC：Chinese medicine bacteria culture preservation administrative center.b)+：Positive findings ,-：Negative findings.

Claims (19)

1. the method for a fast constant temperature detection artificial tuberculosis yersinia genus, it is characterised in that comprise the following steps：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that drawing of artificial tuberculosis yersinia genus genome specificity base sequence can be expanded Thing group, as primer, carries out isothermal amplification reactions under enzyme reaction system；
(3) by judging whether reaction result is positive, determine in testing sample whether there is artificial tuberculosis yersinia genus；
Wherein, described artificial tuberculosis yersinia genus genome specificity base sequence is the pseudoconcretion that No. GI is 153946813 768567～768785bp bit sequence of Ademilson Salmonella genome.
2. the method for claim 1, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specificity The primer sets sequence of base sequence is artificial tuberculosis yersinia genus genome 768567～768785bp that No. GI is 153946813 A part for the nucleotide sequence of position or a part for its complementary strand.
3. method as claimed in claim 2, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specificity The primer sets of base sequence is primer sets A；Or be selected from and wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence Property is 50% and above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’(SEQ ID NO：4).
4. method as claimed in claim 3, it is characterised in that with wall scroll in described primer sets A sequence or its complementary strand sequence Sequence homology is 50% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’(SEQ ID NO：7)；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’(SEQ ID NO：8).
5. method as claimed in claim 2, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specificity The primer sets of base sequence also comprises one or more ring primer；Described ring primer is LF and/or LB.
6. method as claimed in claim 5, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specificity The primer sets of base sequence is selected from following primer sets A ', any one group of B '；Or selected from and described primer sets A ', B ' sequence or its In complementary strand sequence, wall scroll sequence homology is 50% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_A：5’-TATGCTCATCCAGGCGTTTC-3’(SEQ ID NO：9)；
And/or, lower lantern primer LB_A：5’-TGATGGCGATGGGGAAAATT-3’(SEQ ID NO：10)；
Primer sets B '：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_B：5’-GGGATTTCAATATGCTCATCCAG-3’(SEQ ID NO：11)；
And/or, lower lantern primer LB_B：5’-TGATGGCGATGGGGAAAATT-3’(SEQ ID NO：12).
7. the method for claim 1, it is characterised in that in step (2), described enzyme reaction system includes：1×Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, FIP and BIP of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L Primer, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst archaeal dna polymerase, the beet of 0-1.5mol/L Alkali, including or do not include LF and/or the LB primer of 0.4-1.0 μm of ol/L.
8. the method for claim 1, it is characterised in that the response procedures of described isothermal amplification reactions is：1. 60～65 DEG C hatch 10～90min；2. 80 DEG C terminate reaction 2～20min.
9. for the primer in Constant Temperature Detection artificial tuberculosis yersinia genus method as claimed in claim 1, it is characterised in that described Primer includes the primer sets that can expand artificial tuberculosis yersinia genus genome specificity base sequence, and its sequence is No. GI and is A part for the nucleotide sequence of 768567～768785bp position of the artificial tuberculosis yersinia genus genome of 153946813 or it is mutual Mend a part for chain.
10. primer as claimed in claim 9, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specific The primer sets of property base sequence is primer sets A；Or with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence It is 50% and above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’.
11. primers as claimed in claim 10, it is characterised in that single with described primer sets A sequence or its complementary strand sequence Bar sequence homology is 50% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’.
12. primers as claimed in claim 9, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specific The primer sets of property base sequence also comprises one or more ring primer；Described ring primer is LF and/or LB.
13. primers as claimed in claim 12, it is characterised in that described can expand artificial tuberculosis yersinia genus genome specific Property base sequence primer sets be selected from following primer sets A ', any one group of B '；Or selected from and described primer sets A ', B ' sequence or In its complementary strand sequence, wall scroll sequence homology is 50% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-GTTGTTGTATGAATTGCTGG-3’；
Downstream outer primer B3_A：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_A：5’-GTAACGCTGAAATACCGAACTGTAAATGGTTGATGAAGGTGG-3’；
Downstream inner primer BIP_A：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_A：5’-TATGCTCATCCAGGCGTTTC-3’；
And/or, lower lantern primer LB_A：5’-TGATGGCGATGGGGAAAATT-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-GAATTGCTGGAAATGGTTG-3’；
Downstream outer primer B3_B：5’-GCGGATAACTCCTGTTCT-3’；
Upstream inner primer FIP_B：5’-GTAACGCTGAAATACCGAACTGTATGAAGGTGGTTTGAAACG-3’；
Downstream inner primer BIP_B：5’-CACCTTTATCTCTGATAATTTCCGCCTTTCCAGCTCATGTTGAT-3’；
Upper lantern primer LF_B：5’-GGGATTTCAATATGCTCATCCAG-3’；
And/or, lower lantern primer LB_B：5’-TGATGGCGATGGGGAAAATT-3’.
14. 1 kinds of kits for Constant Temperature Detection artificial tuberculosis yersinia genus, it is characterised in that described kit includes such as power Profit requires the primer described in any one of 9～13.
15. kits as claimed in claim 14, it is characterised in that its also include Bst DNA polymerase reaction buffer solution, Bst archaeal dna polymerase, dNTP solution, Mg2+, one or more in glycine betaine.
16. 1 kinds of kits for Constant Temperature Detection artificial tuberculosis yersinia genus, it is characterised in that the enzyme reaction of described kit System includes：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-2.0 μ FIP and the BIP primer of mol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, including or do not include the LF of 0.4-1.0 μm of ol/L And/or LB primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase, and the glycine betaine of 0-1.5mol/L.
17. 1 kinds of carriers, it is characterised in that described carrier comprises the primer as described in any one of claim 9～13.
Application in Constant Temperature Detection artificial tuberculosis yersinia genus for 18. primers, it is characterised in that described primer is for such as claim Primer described in any one of 9～13.
19. kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are in Constant Temperature Detection Application in artificial tuberculosis yersinia genus.
CN201610780457.8A 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature Active CN106434898B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202010036128.9A CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application
CN202010035792.1A CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201510556917 2015-09-02
CN2015105569174 2015-09-02

Related Child Applications (2)

Application Number Title Priority Date Filing Date
CN202010035792.1A Division CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis
CN202010036128.9A Division CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application

Publications (2)

Publication Number Publication Date
CN106434898A true CN106434898A (en) 2017-02-22
CN106434898B CN106434898B (en) 2020-02-21

Family

ID=57899245

Family Applications (54)

Application Number Title Priority Date Filing Date
CN202010035771.XA Active CN111073955B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of vibrio cholerae O1 group and application
CN202010042450.2A Active CN111057781B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer group and application
CN202010036146.7A Active CN111020047B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer set and application
CN202010035792.1A Pending CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis
CN202010035787.0A Active CN111041071B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio cholerae O1 group, primer group and application
CN201610767491.1A Active CN106434885B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio cholerae O1 group at constant temperature, primer and application
CN201610767426.9A Active CN106434884B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting listeria monocytogenes at constant temperature
CN202010003956.2A Pending CN110951840A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method and kit for vibrio cholerae O1 group
CN201610780456.3A Active CN106434897B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio cholerae O1 group at constant temperature
CN202010036128.9A Pending CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application
CN202010035775.8A Active CN111041112B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio vulnificus, primer set and application
CN202010042444.7A Active CN111057779B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for vibrio vulnificus and application
CN202010286794.8A Pending CN111334595A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method
CN202010036105.8A Active CN111020008B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method and kit for nucleic acid of vibrio cholerae O1 group
CN202010042445.1A Active CN111020049B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for staphylococcus aureus
CN202010036143.3A Active CN111020010B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method, primer set and kit for listeria monocytogenes
CN201610780460.XA Active CN106434899B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting bacillus cereus at constant temperature
CN201610767506.4A Active CN106434886B (en) 2015-09-02 2016-08-30 Method for rapidly detecting yersinia pseudotuberculosis at constant temperature, primer and application
CN202010004056.XA Active CN110964788B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of cronobacter sakazakii, primer group and application
CN202010035791.7A Active CN111073956B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio vulnificus
CN202010043524.4A Active CN111041116B (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method and kit for vibrio vulnificus
CN202010004054.0A Active CN110964786B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii
CN202010035788.5A Active CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN201911281772.6A Active CN110938677B (en) 2015-09-02 2016-08-30 Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application
CN201610767389.1A Active CN106434882B (en) 2015-09-02 2016-08-30 Method for rapidly detecting cronobacter sakazakii at constant temperature, primer and application
CN201610767354.8A Active CN106244706B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapid constant temperature detection of cronobacter sakazakii
CN201610767576.XA Active CN106434888B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting staphylococcus aureus at constant temperature
CN202010036127.4A Active CN111020045B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method
CN202010036130.6A Active CN111020009B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN201610767402.3A Active CN106434883B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio vulnificus at constant temperature and application of primer and kit
CN202010035808.9A Active CN111041115B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN201610767436.2A Active CN106367492B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting listeria monocytogenes at constant temperature
CN201911167632.6A Active CN110760569B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii
CN202010036148.6A Active CN111020048B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for shigella
CN201911337926.9A Active CN110951837B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010286793.3A Active CN111304348B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN202010035795.5A Active CN111041114B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN201610780457.8A Active CN106434898B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
CN202010043523.XA Active CN111100939B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of staphylococcus aureus and application thereof
CN201911337898.0A Active CN110938678B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting listeria monocytogenes at constant temperature
CN201610780425.8A Active CN106434895B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio parahaemolyticus at constant temperature
CN202010004057.4A Pending CN110964789A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio cholerae O1 group, primer set and application
CN201610767579.3A Active CN106434889B (en) 2015-09-02 2016-08-30 Method for rapidly detecting bacillus cereus at constant temperature, primers and application
CN202010042440.9A Pending CN111020011A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN202010042446.6A Active CN111057780B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN202010042443.2A Active CN111057778B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio vulnificus and application
CN202010004055.5A Active CN110964787B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for cronobacter sakazakii
CN202010003957.7A Pending CN110951841A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for vibrio cholerae O1 group
CN201911167633.0A Active CN110760570B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapid constant-temperature detection of cronobacter sakazakii
CN202010035812.5A Active CN111073989B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and application of shigella nucleic acid
CN201610780447.4A Active CN106434896B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application
CN201610767703.6A Active CN106434891B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting shigella at constant temperature
CN201911281105.8A Active CN110938676B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
CN201610780421.XA Active CN106367500B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio vulnificus at constant temperature, primer and application

Family Applications Before (37)

Application Number Title Priority Date Filing Date
CN202010035771.XA Active CN111073955B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of vibrio cholerae O1 group and application
CN202010042450.2A Active CN111057781B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer group and application
CN202010036146.7A Active CN111020047B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer set and application
CN202010035792.1A Pending CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis
CN202010035787.0A Active CN111041071B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio cholerae O1 group, primer group and application
CN201610767491.1A Active CN106434885B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio cholerae O1 group at constant temperature, primer and application
CN201610767426.9A Active CN106434884B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting listeria monocytogenes at constant temperature
CN202010003956.2A Pending CN110951840A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method and kit for vibrio cholerae O1 group
CN201610780456.3A Active CN106434897B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio cholerae O1 group at constant temperature
CN202010036128.9A Pending CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application
CN202010035775.8A Active CN111041112B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio vulnificus, primer set and application
CN202010042444.7A Active CN111057779B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for vibrio vulnificus and application
CN202010286794.8A Pending CN111334595A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method
CN202010036105.8A Active CN111020008B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method and kit for nucleic acid of vibrio cholerae O1 group
CN202010042445.1A Active CN111020049B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for staphylococcus aureus
CN202010036143.3A Active CN111020010B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method, primer set and kit for listeria monocytogenes
CN201610780460.XA Active CN106434899B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting bacillus cereus at constant temperature
CN201610767506.4A Active CN106434886B (en) 2015-09-02 2016-08-30 Method for rapidly detecting yersinia pseudotuberculosis at constant temperature, primer and application
CN202010004056.XA Active CN110964788B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of cronobacter sakazakii, primer group and application
CN202010035791.7A Active CN111073956B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio vulnificus
CN202010043524.4A Active CN111041116B (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method and kit for vibrio vulnificus
CN202010004054.0A Active CN110964786B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii
CN202010035788.5A Active CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN201911281772.6A Active CN110938677B (en) 2015-09-02 2016-08-30 Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid and application
CN201610767389.1A Active CN106434882B (en) 2015-09-02 2016-08-30 Method for rapidly detecting cronobacter sakazakii at constant temperature, primer and application
CN201610767354.8A Active CN106244706B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapid constant temperature detection of cronobacter sakazakii
CN201610767576.XA Active CN106434888B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting staphylococcus aureus at constant temperature
CN202010036127.4A Active CN111020045B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for nucleic acid of bacillus cereus and application of rapid constant-temperature detection method
CN202010036130.6A Active CN111020009B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN201610767402.3A Active CN106434883B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio vulnificus at constant temperature and application of primer and kit
CN202010035808.9A Active CN111041115B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN201610767436.2A Active CN106367492B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting listeria monocytogenes at constant temperature
CN201911167632.6A Active CN110760569B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii
CN202010036148.6A Active CN111020048B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for shigella
CN201911337926.9A Active CN110951837B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010286793.3A Active CN111304348B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN202010035795.5A Active CN111041114B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application

Family Applications After (16)

Application Number Title Priority Date Filing Date
CN202010043523.XA Active CN111100939B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of staphylococcus aureus and application thereof
CN201911337898.0A Active CN110938678B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting listeria monocytogenes at constant temperature
CN201610780425.8A Active CN106434895B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting vibrio parahaemolyticus at constant temperature
CN202010004057.4A Pending CN110964789A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio cholerae O1 group, primer set and application
CN201610767579.3A Active CN106434889B (en) 2015-09-02 2016-08-30 Method for rapidly detecting bacillus cereus at constant temperature, primers and application
CN202010042440.9A Pending CN111020011A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN202010042446.6A Active CN111057780B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN202010042443.2A Active CN111057778B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio vulnificus and application
CN202010004055.5A Active CN110964787B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for cronobacter sakazakii
CN202010003957.7A Pending CN110951841A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for vibrio cholerae O1 group
CN201911167633.0A Active CN110760570B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapid constant-temperature detection of cronobacter sakazakii
CN202010035812.5A Active CN111073989B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and application of shigella nucleic acid
CN201610780447.4A Active CN106434896B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application
CN201610767703.6A Active CN106434891B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting shigella at constant temperature
CN201911281105.8A Active CN110938676B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
CN201610780421.XA Active CN106367500B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio vulnificus at constant temperature, primer and application

Country Status (1)

Country Link
CN (54) CN111073955B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114182029A (en) * 2021-11-30 2022-03-15 石家庄君乐宝乳业有限公司 Primer combination and application thereof in detection of cronobacter sakazakii in dairy products

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111073955B (en) * 2015-09-02 2022-10-21 上海产业技术研究院 Rapid isothermal detection method for nucleic acid of vibrio cholerae O1 group and application
CN107058599A (en) * 2017-06-22 2017-08-18 上海速创诊断产品有限公司 A kind of Primer composition, kit and its dual signal channel detection methods for detecting staphylococcus aureus
CN107475401B (en) * 2017-09-08 2021-03-19 江苏农林职业技术学院 Method and primer for detecting food-borne bacillus cereus by using loop-mediated isothermal amplification technology
CN108285925A (en) * 2017-12-29 2018-07-17 广东环凯微生物科技有限公司 A kind of rugged Cronobacter sakazakii quick detection kit of slope
CN108192988B (en) * 2018-03-06 2020-05-19 青岛大学 Staphylococcus aureus strand exchange amplification detection method
CN108611402A (en) * 2018-05-12 2018-10-02 浙江工商大学 Shigella flexneri visible detection method based on aptamers magnetic capture and direct LAMP
CN109680079A (en) * 2018-06-08 2019-04-26 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） Detect RPA primer, probe, kit and the method for vibrio parahemolyticus
CN109593866A (en) * 2018-06-20 2019-04-09 齐鲁工业大学 Primer, kit and the detection method of ring mediated isothermal amplification Listeria monocytogenes
CN109517914A (en) * 2018-12-27 2019-03-26 广东环凯微生物科技有限公司 The dry powdered double PCR detection kit of the rugged Cronobacter sakazakii of slope
CN110257541B (en) * 2019-07-25 2022-08-09 沈阳农业大学 CAMP detection primer group and kit for enterotoxin gene of bacillus cereus
CN110358851B (en) * 2019-08-14 2023-01-17 河南科技学院 Nucleic acid sequence, primer, method and kit for detecting bacillus cereus
CN111172325A (en) * 2020-02-21 2020-05-19 北京天恩泽基因科技有限公司 Multi-target double-dye isothermal amplification rapid detection method and kit
CN111690757A (en) * 2020-05-19 2020-09-22 广东岭南职业技术学院 Primer and detection method for rapidly identifying vomitoxin-producing bacillus cereus
CN112538549A (en) * 2020-12-07 2021-03-23 菲吉乐科(南京)生物科技有限公司 On-site rapid detection test method for phage activity
CN112646908A (en) * 2020-12-31 2021-04-13 广州赛哲生物科技股份有限公司 Vibrio vulnificus isothermal amplification primer, probe, kit and detection method
CN115261493A (en) * 2021-04-30 2022-11-01 上海旺旺食品集团有限公司 Method for detecting cronobacter sakazakii and application thereof
CN113512554B (en) * 2021-07-09 2022-07-12 合肥工业大学 Protein for regulating sakazakii cronobacter sakazakii pressure-resistant strong stress, encoding gene thereof and application thereof
CN113846173A (en) * 2021-09-01 2021-12-28 东北农业大学 Novel target, primer group and detection method for cronobacter sakazakii detection
CN113957164B (en) * 2021-10-29 2023-05-23 上海市质量监督检验技术研究院 CRISPR One post detection method and kit thereof for Cronobacter in infant formula powder
CN114540516B (en) * 2022-03-08 2023-06-20 河南中检食安生物科技有限公司 LAMP double-strand detection probe, kit and detection method for staphylococcus aureus
CN115851996A (en) * 2022-09-30 2023-03-28 上海纳米技术及应用国家工程研究中心有限公司 Preparation method of positive quality control for staphylococcus aureus detection

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182575A (en) * 2007-11-19 2008-05-21 天津出入境检验检疫局动植物与食品检测中心 Method for detecting food-borne pseudotuberculosis yersinia genus by loop-mediated isothermal amplification
CN101492733A (en) * 2008-12-15 2009-07-29 天津出入境检验检疫局动植物与食品检测中心 Reagent kit and method for detection of artificial tuberculosis yersinia genus with ring mediated isothermality amplification method

Family Cites Families (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001288360A1 (en) * 2000-08-23 2002-03-04 Genome Therapeutics Corporation Genomics-assisted rapid identification of targets
US20040029129A1 (en) * 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms
JP2003199572A (en) * 2001-12-28 2003-07-15 Eiken Chem Co Ltd Primer for detection of salmonella and detection method using the same
JP4226984B2 (en) * 2003-09-26 2009-02-18 日本ハム株式会社 LAMP primer for detection of Listeria monocytogenes
JP2007129935A (en) * 2005-11-09 2007-05-31 Ishikawa Pref Gov Primer specifically detecting microorganism in sample
CN101020927A (en) * 2007-03-09 2007-08-22 中国科学院南海海洋研究所 Reagent kit and process for detecting Vibrio vulnificus in circular mediated constant temperature amplification method
CN101153326B (en) * 2007-09-21 2011-03-23 珠海市疾病预防控制中心 Primer, detection method and detection reagent kit for detecting shigella
CN101153332B (en) * 2007-09-21 2011-03-23 珠海市疾病预防控制中心 Primer, detection method and detection reagent kit for detecting cholera vibrio
CN101153330B (en) * 2007-09-21 2011-07-13 珠海市疾病预防控制中心 Primer, detection method and detection reagent kit for detecting vibrio parahemolyticus
CN101153329B (en) * 2007-09-21 2010-11-03 珠海市疾病预防控制中心 Primer, detection method and detection reagent kit for detecting staphylococcus aureus
CN101140243B (en) * 2007-09-29 2010-04-14 上海水产大学 Method for detecting vibrio parahaemolyticus
CN101200760A (en) * 2007-12-13 2008-06-18 中国检验检疫科学研究院 Preparation and utilization method of yersinia genus rapid detection reagent kit
CN101245375A (en) * 2007-12-13 2008-08-20 山东出入境检验检疫局检验检疫技术中心 Method for producing and using trauma vibrio fast detection kit
CN101307351A (en) * 2008-04-29 2008-11-19 广州华峰生物科技有限公司 Rapid diagnosis kit for listeria monocytogenes gene based on loop-mediated isothermal amplification technology and detecting method thereof
CN101319249B (en) * 2008-06-10 2011-05-11 山东出入境检验检疫局检验检疫技术中心 Fast detecting reagent kit for enterobacter sakazakii and detecting method thereof
CN101348835B (en) * 2008-09-09 2011-08-17 南开大学 Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology
CN101368204B (en) * 2008-09-16 2011-08-31 中国计量学院 Fast detection primer and reagent kit for enterobacter sakazakii hymenial veil mediated isothermality amplification technique
CN101403004B (en) * 2008-09-26 2011-08-24 广州华峰生物科技有限公司 Rapid diagnosis reagent kit and detection method for vibrio vulnficus gene
CN101402997B (en) * 2008-11-06 2010-08-11 中华人民共和国天津出入境检验检疫局 Reagent kit and method for detecting bacillus cereus with loop mediated isothermality amplification method
CN101748201B (en) * 2008-11-28 2012-06-27 中华人民共和国黑龙江出入境检验检疫局检验检疫技术中心 Method of loop-mediated isothermal amplification (LAMP) for detecting Listeria monocytogenes
CN101831493B (en) * 2009-11-06 2012-05-23 武汉工业学院 Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method
CN101845493A (en) * 2010-01-29 2010-09-29 华南农业大学 Primer for detection of shigella and detection method
CN101864483B (en) * 2010-04-12 2012-09-19 广州华峰生物科技有限公司 Salmonella and shigella joint detection kit and detection method thereof
CN101824482B (en) * 2010-06-07 2012-09-19 广州华峰生物科技有限公司 Detection kit for vibrio cholerae O1 group and detection method thereof
US10357577B2 (en) * 2010-07-16 2019-07-23 Auckland Uniservices Limited Bacterial nitroreductase enzymes and methods relating thereto
CN102094090B (en) * 2010-12-13 2013-03-13 华东师范大学 Cholera toxin virulence gene detection kit and detection method thereof
CN102154451B (en) * 2010-12-30 2013-07-31 广东省微生物研究所 Loop-mediated isothermal amplification detection primer group, detection method and detection kit for enterobacter sakazakii
CN102206703A (en) * 2011-01-23 2011-10-05 浙江省质量技术监督检测研究院 Multiple rapid detection method for three food borne pathogenic bacteria, and detection primer set and kit thereof
CN102277422A (en) * 2011-06-20 2011-12-14 黑龙江省乳品工业技术开发中心 Method for rapid detection of Listeria monocytogenes viable bacteria in liquid milk
CN102329861B (en) * 2011-08-29 2013-06-05 中国疾病预防控制中心传染病预防控制所 Primer for detecting serotype of shigella flexneri and multiplex amplification using same
US8883488B2 (en) * 2011-11-15 2014-11-11 Tuskegee University Detection of food threat agents and food-borne pathogens
ITMI20112177A1 (en) * 2011-11-29 2013-05-30 Genefast S R L METHOD OF DETECTING SYNTHESIS AND / OR AMPLIFICATION OF A NUCLEIC ACID
CN102719535B (en) * 2012-06-01 2014-02-26 南昌大学 Method for rapidly detecting listeria monocytogenes in food
CN102925588B (en) * 2012-08-02 2014-04-23 四川农业大学 LAMP kit used for rapidly detecting porcine cytomegalovirus
CN102936621B (en) * 2012-08-27 2014-06-11 上海交通大学 Bacillus cereus detection method and kit
CN102864228A (en) * 2012-09-21 2013-01-09 武汉真福医药科技发展有限公司 Loop-mediated isothermal amplification (LAMP) kit for rapidly detecting vibrio parahaemolyticus
CN102851381A (en) * 2012-09-21 2013-01-02 武汉真福医药科技发展有限公司 LAMP kit for rapid detection of Listeria monocytogenes
CN102851382A (en) * 2012-09-21 2013-01-02 武汉真福医药科技发展有限公司 LAMP kit for rapid detection of Shigella
CN103160604A (en) * 2013-04-08 2013-06-19 北京出入境检验检疫局检验检疫技术中心 LAMP (loop-mediated isothermal amplification) detection kit for Vibrio vulnificus and detection method using same
CN103160606B (en) * 2013-04-08 2014-07-30 北京出入境检验检疫局检验检疫技术中心 LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof
CN103243168A (en) * 2013-05-16 2013-08-14 汇智泰康生物技术(北京)有限公司 Kit for detecting vibrio parabaemolyticus in food and using method for kit
CN103243171A (en) * 2013-05-29 2013-08-14 光明乳业股份有限公司 Method for detecting cronobacter sakazakii as well as kit and primer thereof
CN103320435B (en) * 2013-06-28 2015-04-22 华南理工大学 Listeria monocytogenes LAMP (loop-mediated isothermal amplification) detection kit containing internal standard
CN103484536B (en) * 2013-07-10 2015-03-04 东北农业大学 Kit used for rapid detection of enterobacter sakazakii in milk, and applications thereof
CN103421904B (en) * 2013-08-14 2015-04-29 华中农业大学 Listeria monocytogenes LAMP (loop-medicated isothermal amplification) visualized detection method
CN103614466B (en) * 2013-11-11 2015-08-26 宁波大学 The primer detected for the LAMP-LFD of Vibrio vulnificus and probe sequence
CN103571961B (en) * 2013-11-12 2015-04-15 光明乳业股份有限公司 Method, primer pair, target probe, internal standard probe and kit for detecting Cronobacter sakazakii
CN104212885B (en) * 2014-06-26 2016-06-22 舟山出入境检验检疫局综合技术服务中心 The LAMP kit of vibrio cholera in a kind of aquatic products
CN104293954A (en) * 2014-10-13 2015-01-21 河北省食品检验研究院 LAMP primer of staphylococcus aureus and application method of LAMP primer
CN104313173B (en) * 2014-11-11 2016-05-04 舟山市质量技术监督检测研究院 The real-time turbidity LAMP of Listeria Monocytogenes detection method
CN104328208A (en) * 2014-11-24 2015-02-04 武汉明曼基因工程有限公司 Rapid detection kit of Shigella and application of rapid detection kit
CN104513857A (en) * 2014-12-22 2015-04-15 广东省微生物研究所 Loop-mediated isothermal amplification detection primer group, detection method and kit of vibrio parahaemolyticus
CN104911249A (en) * 2014-12-22 2015-09-16 浙江海隆生物科技有限公司 Kit for rapidly detecting staphylococcus aureus in milk animal and raw milk
CN104593516A (en) * 2015-02-09 2015-05-06 江南大学 Isothermal amplification method for rapid detection of listeria monocytogenes
CN104862399B (en) * 2015-05-21 2018-06-19 渤海大学 Detect the PCR method and kit containing amplification interior label of bacillus cereus in food
CN111073955B (en) * 2015-09-02 2022-10-21 上海产业技术研究院 Rapid isothermal detection method for nucleic acid of vibrio cholerae O1 group and application
CN105861702A (en) * 2016-05-16 2016-08-17 昆明理工大学 Specific gene of staphylococcus aureus and loop-mediated isothermal amplification kit

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182575A (en) * 2007-11-19 2008-05-21 天津出入境检验检疫局动植物与食品检测中心 Method for detecting food-borne pseudotuberculosis yersinia genus by loop-mediated isothermal amplification
CN101492733A (en) * 2008-12-15 2009-07-29 天津出入境检验检疫局动植物与食品检测中心 Reagent kit and method for detection of artificial tuberculosis yersinia genus with ring mediated isothermality amplification method

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EPPINGER,M.等: "Yersinia pseudotuberculosis IP 31758,complete genome", 《GENBANK DATABASE》 *
HONGWEI ZHANG等: "Loop-Mediated Isothermal Amplification Assays for Detecting Yersinia pseudotuberculosis in Milk Powders", 《JOURNAL OF FOOD SCIENCE》 *
黄国亮等: "《生物医学检测技术与临床检验》", 30 September 2014, 清华大学出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114182029A (en) * 2021-11-30 2022-03-15 石家庄君乐宝乳业有限公司 Primer combination and application thereof in detection of cronobacter sakazakii in dairy products

Also Published As

Publication number Publication date
CN111073956A (en) 2020-04-28
CN106434884B (en) 2020-02-21
CN106434885A (en) 2017-02-22
CN111020009B (en) 2022-07-22
CN111073989A (en) 2020-04-28
CN106434888A (en) 2017-02-22
CN106434889B (en) 2020-06-09
CN111020048B (en) 2023-03-10
CN106434891A (en) 2017-02-22
CN111100906A (en) 2020-05-05
CN110964788A (en) 2020-04-07
CN106434882B (en) 2020-02-21
CN106434895B (en) 2020-02-21
CN110938676B (en) 2022-07-26
CN111041115B (en) 2022-07-26
CN106434898B (en) 2020-02-21
CN106367492B (en) 2020-02-07
CN111057781A (en) 2020-04-24
CN110760569A (en) 2020-02-07
CN111020047A (en) 2020-04-17
CN106434889A (en) 2017-02-22
CN106434895A (en) 2017-02-22
CN106434886A (en) 2017-02-22
CN110760570B (en) 2023-01-24
CN111020045B (en) 2022-10-21
CN106434896B (en) 2020-02-18
CN111020046A (en) 2020-04-17
CN110760569B (en) 2023-01-24
CN111073989B (en) 2023-05-02
CN111020049B (en) 2022-07-26
CN106434891B (en) 2020-02-18
CN106434899A (en) 2017-02-22
CN111041113A (en) 2020-04-21
CN106367500B (en) 2020-02-21
CN106434882A (en) 2017-02-22
CN106434896A (en) 2017-02-22
CN111020009A (en) 2020-04-17
CN106434883A (en) 2017-02-22
CN111057778A (en) 2020-04-24
CN111334595A (en) 2020-06-26
CN111041071B (en) 2022-10-21
CN111041115A (en) 2020-04-21
CN106244706B (en) 2020-01-10
CN111041071A (en) 2020-04-21
CN111041112B (en) 2022-09-20
CN110938678B (en) 2022-08-26
CN111057780B (en) 2022-07-22
CN110964788B (en) 2022-08-26
CN110938677A (en) 2020-03-31
CN111020008A (en) 2020-04-17
CN106367492A (en) 2017-02-01
CN106367500A (en) 2017-02-01
CN110964786B (en) 2022-08-26
CN110964787A (en) 2020-04-07
CN110951841A (en) 2020-04-03
CN110938678A (en) 2020-03-31
CN106434886B (en) 2020-01-21
CN110760570A (en) 2020-02-07
CN106434897B (en) 2020-02-21
CN111041116B (en) 2022-07-22
CN111057779B (en) 2022-08-26
CN110951837A (en) 2020-04-03
CN111057778B (en) 2022-07-26
CN110951837B (en) 2022-08-26
CN111020048A (en) 2020-04-17
CN111041113B (en) 2022-10-21
CN106434888B (en) 2020-02-21
CN111020010B (en) 2023-05-02
CN111041112A (en) 2020-04-21
CN111057779A (en) 2020-04-24
CN110951840A (en) 2020-04-03
CN106434884A (en) 2017-02-22
CN111020049A (en) 2020-04-17
CN106244706A (en) 2016-12-21
CN111057781B (en) 2022-07-26
CN111304348A (en) 2020-06-19
CN111073955B (en) 2022-10-21
CN110964787B (en) 2022-08-26
CN111020045A (en) 2020-04-17
CN111304348B (en) 2022-07-26
CN106434897A (en) 2017-02-22
CN106434899B (en) 2020-02-21
CN111041116A (en) 2020-04-21
CN111020047B (en) 2022-07-26
CN106434883B (en) 2020-02-21
CN110938677B (en) 2022-07-22
CN111020010A (en) 2020-04-17
CN111073956B (en) 2022-09-20
CN110964789A (en) 2020-04-07
CN111100939A (en) 2020-05-05
CN111057780A (en) 2020-04-24
CN110964786A (en) 2020-04-07
CN110938676A (en) 2020-03-31
CN111041114A (en) 2020-04-21
CN111041114B (en) 2023-03-28
CN111100939B (en) 2022-07-26
CN111020008B (en) 2022-10-28
CN111020011A (en) 2020-04-17
CN111073955A (en) 2020-04-28
CN106434885B (en) 2020-02-14

Similar Documents

Publication Publication Date Title
CN106434898A (en) Method, primers and kit for quickly detecting yersinia pseudotuberculosis at constant temperature
CN106367493A (en) Method for rapidly detecting salmonella at constant temperature, primer and applications of primer
CN106434890A (en) Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner
CN106434887A (en) Method, primers and kit for rapid constant-temperature detection of staphylococcus aureus
CN106367501A (en) Method for rapidly detecting salmonella at constant temperature, primer and kit
CN106434900A (en) Method for conducting rapid constant-temperature detection on vibrio vulnificus and vibrio cholerae simultaneously, primer and kit
CN106367499A (en) Method for rapidly detecting vibrio vulnificus at constant temperature, primer and kit

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant