CN106434899A - Method, primers and kit for quickly detecting bacillus cereus at constant temperature - Google Patents

Method, primers and kit for quickly detecting bacillus cereus at constant temperature Download PDF

Info

Publication number
CN106434899A
CN106434899A CN201610780460.XA CN201610780460A CN106434899A CN 106434899 A CN106434899 A CN 106434899A CN 201610780460 A CN201610780460 A CN 201610780460A CN 106434899 A CN106434899 A CN 106434899A
Authority
CN
China
Prior art keywords
primer
bacillus cereus
sequence
primer sets
sets
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
CN201610780460.XA
Other languages
Chinese (zh)
Other versions
CN106434899B (en
Inventor
贾犇
李雪玲
刘伟
韦朝春
李园园
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 CN202010035788.5A priority Critical patent/CN111041113B/en
Priority to CN202010036127.4A priority patent/CN111020045B/en
Publication of CN106434899A publication Critical patent/CN106434899A/en
Application granted granted Critical
Publication of CN106434899B publication Critical patent/CN106434899B/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 bacillus cereus 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 under an enzyme reaction system by taking the genomic DNA as a template and taking the primer group capable of amplifying specific sequences of the bacillus cereus as the primers; whether the bacillus cereus 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 the high specificity are achieved, the detection time is short, result determination is easy, operation is convenient and fast, the cost is low, and a wide application prospect is achieved.

Description

The method of fast constant temperature detection bacillus cereus, primer and kit
Technical field
The invention belongs to biological technical field, be specifically related to the method for a kind of fast constant temperature detection bacillus cereus, draw Thing and kit.
Background technology
Bacillus cereus (Bacillus cereus) is distributed widely in nature, is that a kind of common food-borne causes Sick bacterium.Clinically, the food poisoning that bacillus cereus causes can be divided into diarrhea-type and vomiting type two types, is respectively Caused by the diarrhoea toxin being produced by this bacterium and vomitoxin, the former is sensitive to heat and trypsase, thus can prevent；And the latter couple Thermally-stabilised, activity is not by pepsic interference, even if trace expression also can have bigger harm to human body, is difficult to take precautions against, Chang Yin Play fulminant food poisoning.Therefore, for the prevention of this bacterium with detect extremely important.
Tradition bacillus cereus detection method is longer due to the detection cycle, operates relative complex, and detection efficiency is relatively low, difficult With meet modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, want easily Ask.Recently as the development of nucleic acid molecules detection technique, researcher have also been developed the detection means such as PCR, but the party Method needs special detecting instrument, therefore, is not appropriate for being widely used in inside detection department of basic unit especially enterprise's production line The real-time on-site detection carrying out.In order to ensure food security, be badly in need of quick, simple, accurately method detect the wax in food Sample bacillus.
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.Bacillus cereus contains plasmid, therefore prior art such as patent of invention CN 101831493 B and CN 101402997 B are the design carrying out bacillus cereus LAMP primer based on target sequence on plasmid or chromosome respectively.But, Owing to plasmid exists unstability on copy number and genetic structure, and for for bacillus cereus dyeing in prior art The LAMP primer of the target sequence design on body there is also the inadequate problem of primer specificity, illustrates invention in table 1 of the present invention This problem that patent CN 101402997 B exists.It is to say, the bacillus cereus used in art methods Detection sequence actually not bacillus cereus institute is peculiar, i.e. be possible to non-bacillus cereus is regarded as wax mistakenly Sample bacillus.Therefore, need one in industry badly and be able to ensure that specific bacillus cereus detection method, meet base simultaneously Layer detection department, to quick, 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 bacillus cereus, and forms Gao Ling on this basis Sensitivity, high specific detection kit.The present invention based in GenBank database microbial genome data resource (by On August 5th, 2013 data) carry out the design of bacillus cereus LAMP primer, provide a kind of fast constant temperature augmentation detection wax The method of sample bacillus, primer sets and kit.Use the detection method detection bacillus cereus of the present invention, there is Gao Ling Sensitivity and high specific, the detection time is short, and result judges simple, simple operation, the advantage of low cost.
The present invention proposes a kind of method of quick detection Bacillus cereus strain, said method comprising the steps of：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that drawing of bacillus cereus genome specificity base sequence can be expanded Thing group is primer, under enzyme reaction system, carries out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine in testing sample whether there is bacillus cereus.
The method of Constant Temperature Detection Bacillus cereus strain of the present invention, extracts genomic DNA, which is from testing sample Template, with bacillus cereus specificity amplification primer group as primer, carries out isothermal amplification reactions, then, by judging to react Whether result is positive, determines in testing sample whether there is bacillus cereus.Wherein, described enzyme reaction system include but not It is limited to DNA polymerase reaction system.
In the present invention, described bacillus cereus genome specificity base sequence be No. GI be 218895141 waxy 3193434～3194011bp bit sequence of bacillus.
In the present invention, the described primer sets that can expand bacillus cereus genome specificity base sequence is described gene A part for the nucleotide sequence of 3193434～3194011bp position of group (No. GI is 218895141) or one of its complementary strand Point.Wherein, described bacillus cereus genome specificity base sequence refers to that only bacillus cereus genome institute is peculiar , and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand bacillus cereus genome specific base sequence includes but is not limited to primer Group A, or be 55% and above primer sets with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence.
Primer sets A：
Upstream outer primer F3_A：5’-AGGAACTATTAGAAAACGCG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’ (SEQ ID NO：4).
In the present invention, the described primer sets that can expand bacillus cereus genome specific base sequence can also include with In aforementioned each primer sets sequence or its complementary strand sequence, wall scroll sequence homology is 55% and above primer sets, this primer sets bag Include but be not limited to following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-TAGAAAACGCGGGTATGA-3’(SEQ ID NO：5) (with primers F 3_A 5 '- AGGAACTATTAGAAAACGCG-3 ' homology is 55%)；
Downstream outer primer B3_B：5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’(SEQ ID NO：7)；
Downstream inner primer BIP_B：5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’ (SEQ ID NO：8).
In the inventive method, in one embodiment, the enzyme reaction system of described constant-temperature amplification is：1×Bst DNA Polymeric enzyme reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L FIP and BIP 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 and 0- 1.5mol/L glycine betaine.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, Comprise 20mmol/L Tris-HCl (pH 8.8), 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 bacillus cereus is positive, containing waxy gemma Bacillus；Band as stepped in electrophoretogram not expression characteristics, then testing sample is that bacillus cereus is negative.Described turbidity is examined Surveying, being to detect by an unaided eye or transmissometer detection turbidity, detection pipe occurs substantially muddy, then testing sample is bacillus cereus sun Property, containing bacillus cereus；As muddy in having no, then testing sample is that bacillus cereus is negative.Also can after centrifugal meat Whether eye is observed has precipitation at the bottom of reaction tube, if there being precipitation at the bottom of reaction tube, then testing sample is that bacillus cereus is positive, containing wax Sample bacillus；Do not precipitate as at the bottom of reaction tube, then testing sample is that bacillus cereus is negative.
Described color developing detection, is addition developer, including but not limited to calcein (50 μM) or SYBR in reaction tube Green I (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 bacillus cereus is negative；As after reaction, color is green Look, then testing sample is that bacillus cereus is positive, containing bacillus cereus.When employing hydroxynaphthol blue is as developer When, as after reaction, color is pansy, then testing sample is that bacillus cereus is negative；As after reaction, color is sky blue, Then testing sample is that bacillus cereus is positive.Described color developing detection, in addition to above by visually observing reaction result, it is possible to To carry out in real time or end point determination reaction result by detecting instrument, by the rational threshold value setting negative reaction, when to be measured When the result of example reaction is less than or equal to this threshold value, then testing sample is that bacillus cereus is negative；When testing sample reaction Result more than this threshold value when, then testing sample is that bacillus cereus is positive.Described detecting instrument includes but is not limited to fluorescence Spectrophotometer, quantitative real time PCR Instrument, constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument and the inspection of Genie II isothermal duplication fluorescence Examining 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 Bacillus cereus strain.Described primer includes Can expand the primer sets of bacillus cereus genome specific base sequence, it includes but is not limited to, and the sequence of described primer is No. GI be 218895141 bacillus cereus genome 3193434～3194011bp position nucleotide sequence a part or A part for its complementary strand.
Wherein, the described primer sets that can expand bacillus cereus genome specificity base sequence is selected from following primer Any one group of group, or selected from wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence be 55% and with On arbitrary primer sets.Wherein, described primer sets includes but is not limited to following primer sets A.Described with aforementioned primer sets sequence or In its complementary strand sequence, wall scroll sequence homology is 55% and above primer sets includes but is not limited to following primer sets B.
Primer sets A：
Upstream outer primer F3_A：5’-AGGAACTATTAGAAAACGCG-3’；
Downstream outer primer B3_A：5’-CCGGTTTAGATAATTCACGT-3’；
Upstream inner primer FIP_A：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’；
Downstream inner primer BIP_A：5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’；
Primer sets B：
Upstream outer primer F3_B：5’-TAGAAAACGCGGGTATGA-3’；
Downstream outer primer B3_B：5’-CCGGTTTAGATAATTCACGT-3’；
Upstream inner primer FIP_B：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’；
Downstream inner primer BIP_B：5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’.
The present invention also provides a kind of for the kit in above-mentioned Constant Temperature Detection Bacillus cereus strain method, and it includes The described primer sets that can expand bacillus cereus genome specific base sequence.In kit of the present invention, described can expand wax The primer sets of sample bacillus gene group-specific base sequence, including but not limited to genome (No. GI:218895141) A part for a part for the nucleotide sequence of 3193434～3194011bp position or its complementary strand is as described primer sequence；Described Primer includes but is not limited to described primer sets A.Also including but not limited to single with aforementioned primer sequence or its complementary strand sequence Bar sequence homology is 55% and above primer sets is as primer；Including but not limited to primer sets B.
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/L FIP and the BIP primer of dNTP, 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 Archaeal 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%Triton X-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 bacillus cereus genomic DNA, or comprise waxy gemma bar Bacterium genomic DNA 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 any one group of primer selected from primer sets A, B.This carrier Owing to containing, there is the specific DNA sequence dna of bacillus cereus, therefore can be applicable to microbial taxonomy, Comparative genomic strategy The research fields such as, evolution, and the application such as microorganism detection.This carrier can be but not limited to plasmid vector (as PBR322, pUC18, pUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda etc.) and artificial coloring Body carrier (such as Bacterial artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, any one that comprises primer sets A is drawn The carrier pBR322-A of thing, the carrier pBR322-B etc. comprising any one primer of primer sets B.Comprise primer sets A arbitrarily Article one, carrier bacteriophage lambda-A, the carrier bacteriophage lambda-B etc. comprising any one primer of primer sets B of primer.
The invention allows for selected from primer sets A, the primer of any one group of B in Constant Temperature Detection bacillus cereus Application.
The invention allows for application in Constant Temperature Detection bacillus cereus for the described kit.
The invention allows for application in Constant Temperature Detection bacillus cereus for the described carrier.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection bacillus cereus Method, primer/primer sets, detection reagent/kit, greater significance is had to the food security of China.The beneficial effect of the present invention Fruit includes：Use that bacillus cereus detection method of the present invention has high specificity, highly sensitive, the detection time is short, result is sentenced Calmly simply, simple operation, low cost and other advantages.Compared with conventional at present detection method, the constant-temperature amplification method that the present invention uses, can To carry out under constant temperature, only simple thermostat need to be used, it is not necessary to the expensive instrument in PCR experiment, it is not necessary to right Amplified production carries out the steps such as electrophoresis detection, thus, it is very suitable for being widely used in various circles of society and include basic unit's food security inspection Survey department promotes the use of, even if also can fully answer in the environment of molecular biology professional knowledge and skills base relative deficiency With.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 bacillus cereus Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 bacillus cereus 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 bacillus cereus isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Bacillus cereus bacterial classification for detection derives from China General Microbiological DSMZ, numbering CGMCC 1.3760 (=ATCC 14579).Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract examination Agent box extracts genomic DNA, DNA OD260/OD280Being 1.8, concentration is 5.36ng/ μ L.
(2) it with bacillus cereus genomic DNA to be measured as template, is respectively adopted the kit (being shown in Table 2, table 3) of autogamy, And according to condition described in table 3, prepare reaction system, with bacillus cereus specificity amplification primer group as primer, carry out perseverance Isothermal amplification reaction.Primer in embodiment 1～6 is respectively primer sets A, A, A, B, B, B.
(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 Bacillus cereus specific fragment carries out expanding and obtains testing result.Therefore, present invention could apply in detection sample Whether contain bacillus cereus.
Embodiment 7 bacillus cereus specific detection
Collect non-bacillus cereus 28 strain (in table 4 and Fig. 1 1～4,6～29), by these bacterial strains and waxy gemma bar Bacteria strain (in table 4 and Fig. 1 5) is cultivated respectively, takes 1mL bacterium solution, uses kit IA, extracts DNA of bacteria, and with reference in fact Execute reaction system and the condition of example 1, carry out LAMP amplification (primer sets is A) respectively and add developer to observe.
Its testing result is as shown in table 4 and Fig. 1, and in Fig. 1,1～4 is respectively staphylococcus aureus, Staphylococcus aureus The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, 6～29 are respectively gill fungus sample bacillus, listeria monocytogenes, English Promise gram Listeria, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, B-mode pair Salmonella typhi, shigella dysenteriae, Shigella bogdii, shigella flexneri, ETEC (A containing clostridium botulinum Type gene), pathogenic ETEC, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic large intestine Escherichia, hemorrhagic ETEC, yersinia enterocolitica, artificial tuberculosis yersinia genus, Vibrio vulnificus, pair Hemolysis vibrion, Freund vibrios and bacillus cereus, NTC：Negative control, 5：Bacillus cereus.In Fig. 1, only waxy bud Product after spore bacillus strain amplified reaction is rendered as bright green, is positive findings, as shown in No. 5 pipe.And other are non-waxy Product after Bacillus strain and negative control amplified reaction is all rendered as orange, be negative findings, as the 1st～No. 4,6～ Shown in No. 29 pipes and 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 waxy gemma bar Bacteria strain is specific, i.e. the only Bacillus cereus strain amplification positive, and other non-Bacillus cereus strain are feminine gender.
Preparation detection kit, the primer using in kit is primer sets B, by above-mentioned method for detecting specificity, obtains Same testing result, i.e. the product after non-Bacillus cereus strain and negative control amplified reaction is negative findings, waxy Product after Bacillus strain amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively theory analysis is specifically carried out to primer sets A～B, it was found that In the case that each bar primer at most allows three mispairing, each primer sets is up to 1 primer comparison to non-bacillus cereus On, show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
As described in Example 2 extract bacterium CGMCC 1.3760 DNA, use kit IIB, and according to 1ng, 100pg, 10pg, 1pg, 100fg, 10fg DNA ladder degree adds reaction system, and other reaction conditions are with reference to the side of table 3 embodiment 2 Method carries out LAMP amplification (primer sets is A) respectively and adds developer to observe.As in figure 2 it is shown, 1-6 be respectively 1ng, 100pg, 10pg, 1pg, 100fg and 10fg, NTC：Negative control.The product that in Fig. 2,1ng, 100pg and 10pg are processed is rendered as bright Green, is positive findings, and 1pg, 100fg and 10fg are processed and the product of negative control is rendered as orange, is negative findings. Testing result shows, still can be detected in each reaction tube during minimum DNA containing 10pg (being approximately equivalent to 1700 bacteriums).
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, the DNA of as little as 10pg in each reaction tube Still can be detected.
The specific analysis of primer in the existing detection method of table 1 bacillus cereus
Note：The detection regional sequence of primer in patent CN 101402997 B is carried out in common data base resource Blast comparison, determine this detection region respectively with bacillus cereus and each bar primer region mispairing of non-bacillus cereus Base number.If primer region and non-bacillus cereus (including artificial tuberculosis yersinia genus and Yersinia pestis) bacterial strain Base mismatch number is fewer, shows that its matching degree is higher, specifically poorer.
The kit species of table 2 Constant Temperature Detection bacillus cereus and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention bacillus cereus and testing result
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 (15)

1. the method for a fast constant temperature detection bacillus cereus, 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 the primer sets of bacillus cereus genome specificity base sequence can be expanded As 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 bacillus cereus；
Wherein, described bacillus cereus genome specificity base sequence is the bacillus cereus that No. GI is 218895141 3193434～3194011bp bit sequence of genome.
2. the method for claim 1, it is characterised in that described can expand bacillus cereus genome specificity base The primer sets sequence of sequence is the core of bacillus cereus genome 3193434～3194011bp position that No. GI is 218895141 A part for acid sequence or a part for its complementary strand.
3. method as claimed in claim 2, it is characterised in that described can expand bacillus cereus genome specificity base The primer sets of sequence is primer sets A；Or be 55% with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence And any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-AGGAACTATTAGAAAACGCG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-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 55% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-TAGAAAACGCGGGTATGA-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-CCGGTTTAGATAATTCACGT-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’(SEQ ID NO： 7)；
Downstream inner primer BIP_B：5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’(SEQ ID NO：8).
5. 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.
6. 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.
7. for the primer in Constant Temperature Detection bacillus cereus method as claimed in claim 1, it is characterised in that described primer Including the primer sets of bacillus cereus genome specificity base sequence can be expanded, it is 218895141 that its sequence is No. GI A part for the nucleotide sequence of 3193434～3194011bp position of bacillus cereus genome or a part for its complementary strand.
8. primer as claimed in claim 7, it is characterised in that described can expand bacillus cereus genome specificity base The primer sets of sequence is primer sets A；Or be 55% with wall scroll sequence homology in described primer sets A sequence or its complementary strand sequence And any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-AGGAACTATTAGAAAACGCG-3’；
Downstream outer primer B3_A：5’-CCGGTTTAGATAATTCACGT-3’；
Upstream inner primer FIP_A：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTTC-3’；
Downstream inner primer BIP_A：5’-CCACAGAACACAAATGTAACTCATGAATGTTTCTCTCCCTTTCCT-3’.
9. primer as claimed in claim 8, it is characterised in that with wall scroll in described primer sets A sequence or its complementary strand sequence Sequence homology is 55% and above primer sets includes primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-TAGAAAACGCGGGTATGA-3’；
Downstream outer primer B3_B：5’-CCGGTTTAGATAATTCACGT-3’；
Upstream inner primer FIP_B：5’-CTAGCGCCTTTGTATGTTCCTCGCAGAGGTTTTAGAAGTT-3’；
Downstream inner primer BIP_B：5’-CCACAGAACACAAATGTAACTCATGGTTTCTCTCCCTTTCCTTTT-3’.
10. the kit for Constant Temperature Detection bacillus cereus, it is characterised in that described kit includes that right such as is wanted Seek the primer described in any one of 7～9.
11. kits as claimed in claim 10, 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.
12. 1 kinds of kits for Constant Temperature Detection bacillus cereus, it is characterised in that the enzyme reaction system of described kit Including：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/ FIP and the BIP primer of L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst archaeal dna polymerase, and 0- The glycine betaine of 1.5mol/L.
13. 1 kinds of carriers, it is characterised in that described carrier comprises the primer as described in any one of claim 7～9.
Application in Constant Temperature Detection bacillus cereus for 14. primers, it is characterised in that described primer is for such as claim 7～9 The primer described in any one.
15. kits as described in any one of claim 10～12 or carrier as claimed in claim 13 are in Constant Temperature Detection Application in bacillus cereus.
CN201610780460.XA 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting bacillus cereus at constant temperature Active CN106434899B (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202010035788.5A CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN202010036127.4A 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

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
CN202010036127.4A Division 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
CN202010035788.5A Division CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus

Publications (2)

Publication Number Publication Date
CN106434899A true CN106434899A (en) 2017-02-22
CN106434899B CN106434899B (en) 2020-02-21

Family

ID=57899245

Family Applications (54)

Application Number Title Priority Date Filing Date
CN202010035808.9A Active CN111041115B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN201911281772.6A Active CN110938677B (en) 2015-09-02 2016-08-30 Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid 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
CN201610767506.4A Active CN106434886B (en) 2015-09-02 2016-08-30 Method for rapidly detecting yersinia pseudotuberculosis at constant temperature, primer and application
CN202010043524.4A Active CN111041116B (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method and kit for vibrio vulnificus
CN202010035788.5A Active CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
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
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
CN202010042445.1A Active CN111020049B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for staphylococcus aureus
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
CN201610780460.XA Active CN106434899B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting bacillus cereus at constant temperature
CN201610767436.2A Active CN106367492B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting listeria monocytogenes at constant temperature
CN202010035791.7A Active CN111073956B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio vulnificus
CN202010004056.XA Active CN110964788B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of cronobacter sakazakii, primer group and application
CN202010004054.0A Active CN110964786B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii
CN202010036146.7A Active CN111020047B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer set and application
CN202010003956.2A Pending CN110951840A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method and kit for vibrio cholerae O1 group
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
CN202010035795.5A Active CN111041114B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010036128.9A Pending CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application
CN202010042446.6A Active CN111057780B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
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
CN201610767703.6A Active CN106434891B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting shigella at constant temperature
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
CN202010286793.3A Active CN111304348B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN201610780447.4A Active CN106434896B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application
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
CN202010035812.5A Active CN111073989B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and application of shigella nucleic acid
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
CN202010042444.7A Active CN111057779B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for vibrio vulnificus and application
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
CN202010042440.9A Pending CN111020011A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN202010036148.6A Active CN111020048B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for shigella
CN202010043523.XA Active CN111100939B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of staphylococcus aureus and application thereof
CN201911167633.0A Active CN110760570B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapid constant-temperature detection of cronobacter sakazakii
CN201610780421.XA Active CN106367500B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio vulnificus at constant temperature, primer 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
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
CN201610767579.3A Active CN106434889B (en) 2015-09-02 2016-08-30 Method for rapidly detecting bacillus cereus at constant temperature, primers 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
CN201911337926.9A Active CN110951837B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010004055.5A Active CN110964787B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for cronobacter sakazakii
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
CN202010036143.3A Active CN111020010B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method, primer set and kit for listeria monocytogenes
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
CN201911167632.6A Active CN110760569B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii
CN202010036130.6A Active CN111020009B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus 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
CN202010035792.1A Pending CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis
CN201911281105.8A Active CN110938676B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
CN202010042443.2A Active CN111057778B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio vulnificus and application

Family Applications Before (12)

Application Number Title Priority Date Filing Date
CN202010035808.9A Active CN111041115B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
CN201911281772.6A Active CN110938677B (en) 2015-09-02 2016-08-30 Quick constant-temperature detection method for yersinia pseudotuberculosis nucleic acid 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
CN201610767506.4A Active CN106434886B (en) 2015-09-02 2016-08-30 Method for rapidly detecting yersinia pseudotuberculosis at constant temperature, primer and application
CN202010043524.4A Active CN111041116B (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method and kit for vibrio vulnificus
CN202010035788.5A Active CN111041113B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
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
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
CN202010042445.1A Active CN111020049B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for staphylococcus aureus
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

Family Applications After (41)

Application Number Title Priority Date Filing Date
CN201610767436.2A Active CN106367492B (en) 2015-09-02 2016-08-30 Method, primer and application for rapidly detecting listeria monocytogenes at constant temperature
CN202010035791.7A Active CN111073956B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio vulnificus
CN202010004056.XA Active CN110964788B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of cronobacter sakazakii, primer group and application
CN202010004054.0A Active CN110964786B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for cronobacter sakazakii
CN202010036146.7A Active CN111020047B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method of vibrio parahaemolyticus, primer set and application
CN202010003956.2A Pending CN110951840A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method and kit for vibrio cholerae O1 group
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
CN202010035795.5A Active CN111041114B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010036128.9A Pending CN111020046A (en) 2015-09-02 2016-08-30 Nucleic acid rapid constant temperature detection method for yersinia pseudotuberculosis and application
CN202010042446.6A Active CN111057780B (en) 2015-09-02 2016-08-30 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
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
CN201610767703.6A Active CN106434891B (en) 2015-09-02 2016-08-30 Method, primer and kit for rapidly detecting shigella at constant temperature
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
CN202010286793.3A Active CN111304348B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for bacillus cereus
CN201610780447.4A Active CN106434896B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio parahaemolyticus at constant temperature, primer and application
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
CN202010035812.5A Active CN111073989B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and application of shigella nucleic acid
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
CN202010042444.7A Active CN111057779B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for vibrio vulnificus and application
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
CN202010042440.9A Pending CN111020011A (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus and application
CN202010036148.6A Active CN111020048B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for shigella
CN202010043523.XA Active CN111100939B (en) 2015-09-02 2016-08-30 Rapid isothermal detection method for nucleic acid of staphylococcus aureus and application thereof
CN201911167633.0A Active CN110760570B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapid constant-temperature detection of cronobacter sakazakii
CN201610780421.XA Active CN106367500B (en) 2015-09-02 2016-08-30 Method for rapidly detecting vibrio vulnificus at constant temperature, primer 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
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
CN201610767579.3A Active CN106434889B (en) 2015-09-02 2016-08-30 Method for rapidly detecting bacillus cereus at constant temperature, primers 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
CN201911337926.9A Active CN110951837B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method for Listeria monocytogenes nucleic acid and application
CN202010004055.5A Active CN110964787B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for cronobacter sakazakii
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
CN202010036143.3A Active CN111020010B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method, primer set and kit for listeria monocytogenes
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
CN201911167632.6A Active CN110760569B (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii
CN202010036130.6A Active CN111020009B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio parahaemolyticus 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
CN202010035792.1A Pending CN111100906A (en) 2015-09-02 2016-08-30 Rapid constant-temperature detection method, primer group and kit for yersinia pseudotuberculosis
CN201911281105.8A Active CN110938676B (en) 2015-09-02 2016-08-30 Method, primer group and kit for rapidly detecting yersinia pseudotuberculosis at constant temperature
CN202010042443.2A Active CN111057778B (en) 2015-09-02 2016-08-30 Rapid constant temperature detection method for nucleic acid of vibrio vulnificus and application

Country Status (1)

Country Link
CN (54) CN111041115B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257541A (en) * 2019-07-25 2019-09-20 沈阳农业大学 The CAMP detection primer group and kit of bacillus cereus enterotoxin genes
CN110358851A (en) * 2019-08-14 2019-10-22 河南科技学院 For detecting nucleic acid sequence, primer and the method and kit of bacillus cereus

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111041115B (en) * 2015-09-02 2022-07-26 上海产业技术研究院 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
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
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
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
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
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

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101402997A (en) * 2008-11-06 2009-04-08 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 ring mediated isothermality amplification method
CN101831493A (en) * 2009-11-06 2010-09-15 武汉工业学院 Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method

Family Cites Families (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016940A2 (en) * 2000-08-23 2002-02-28 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
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
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
CN101140243B (en) * 2007-09-29 2010-04-14 上海水产大学 Method for detecting vibrio parahaemolyticus
CN101182575B (en) * 2007-11-19 2011-03-23 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
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
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
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
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
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
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
EP2593796A4 (en) * 2010-07-16 2016-07-27 Auckland Uniservices Ltd 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
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
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
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
CN111041115B (en) * 2015-09-02 2022-07-26 上海产业技术研究院 Rapid isothermal nucleic acid detection method and kit for vibrio parahaemolyticus
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
CN101402997A (en) * 2008-11-06 2009-04-08 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 ring mediated isothermality amplification method
CN101831493A (en) * 2009-11-06 2010-09-15 武汉工业学院 Loop-mediated isothermal amplification (LAMP) primer pair of bacillus cereus and detection method

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DODSON,R.J.等: "Bacillus cereus G9842,complete genome", 《GENBANK DATABASE》 *
周巍等: "环介导等温扩增技术检测酸乳中蜡样芽孢杆菌", 《乳业科学与技术》 *
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 (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110257541A (en) * 2019-07-25 2019-09-20 沈阳农业大学 The CAMP detection primer group and kit of bacillus cereus enterotoxin genes
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
CN110358851A (en) * 2019-08-14 2019-10-22 河南科技学院 For detecting nucleic acid sequence, primer and the method and kit 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

Also Published As

Publication number Publication date
CN106434882A (en) 2017-02-22
CN111057781B (en) 2022-07-26
CN111041114A (en) 2020-04-21
CN111073989B (en) 2023-05-02
CN111041113A (en) 2020-04-21
CN106434897A (en) 2017-02-22
CN106434899B (en) 2020-02-21
CN106434888A (en) 2017-02-22
CN111020010B (en) 2023-05-02
CN106434897B (en) 2020-02-21
CN111100906A (en) 2020-05-05
CN111020008A (en) 2020-04-17
CN106434885A (en) 2017-02-22
CN111020045B (en) 2022-10-21
CN106434884A (en) 2017-02-22
CN111057780A (en) 2020-04-24
CN106434883B (en) 2020-02-21
CN111041116B (en) 2022-07-22
CN110951840A (en) 2020-04-03
CN106434889B (en) 2020-06-09
CN106434895B (en) 2020-02-21
CN106434886A (en) 2017-02-22
CN106434888B (en) 2020-02-21
CN106434891A (en) 2017-02-22
CN110964788B (en) 2022-08-26
CN111100939A (en) 2020-05-05
CN111041071A (en) 2020-04-21
CN111041112B (en) 2022-09-20
CN111020009B (en) 2022-07-22
CN111073989A (en) 2020-04-28
CN110760569A (en) 2020-02-07
CN111041113B (en) 2022-10-21
CN110938677A (en) 2020-03-31
CN111020047B (en) 2022-07-26
CN110938677B (en) 2022-07-22
CN110760570A (en) 2020-02-07
CN111334595A (en) 2020-06-26
CN111057778B (en) 2022-07-26
CN111304348A (en) 2020-06-19
CN111041071B (en) 2022-10-21
CN110938678A (en) 2020-03-31
CN110964787A (en) 2020-04-07
CN106434896A (en) 2017-02-22
CN111020009A (en) 2020-04-17
CN111073955A (en) 2020-04-28
CN106434882B (en) 2020-02-21
CN106434891B (en) 2020-02-18
CN111041116A (en) 2020-04-21
CN106367500B (en) 2020-02-21
CN111041115A (en) 2020-04-21
CN106244706A (en) 2016-12-21
CN111020048B (en) 2023-03-10
CN111057781A (en) 2020-04-24
CN111041112A (en) 2020-04-21
CN110938678B (en) 2022-08-26
CN110938676B (en) 2022-07-26
CN106244706B (en) 2020-01-10
CN110964789A (en) 2020-04-07
CN111073955B (en) 2022-10-21
CN111020045A (en) 2020-04-17
CN106434886B (en) 2020-01-21
CN111020010A (en) 2020-04-17
CN111073956B (en) 2022-09-20
CN111020049B (en) 2022-07-26
CN111041115B (en) 2022-07-26
CN110951841A (en) 2020-04-03
CN111057778A (en) 2020-04-24
CN111073956A (en) 2020-04-28
CN106367492A (en) 2017-02-01
CN106367500A (en) 2017-02-01
CN111057779B (en) 2022-08-26
CN110760569B (en) 2023-01-24
CN111020048A (en) 2020-04-17
CN106434885B (en) 2020-02-14
CN111057779A (en) 2020-04-24
CN106434898A (en) 2017-02-22
CN110938676A (en) 2020-03-31
CN111020046A (en) 2020-04-17
CN111041114B (en) 2023-03-28
CN111100939B (en) 2022-07-26
CN111057780B (en) 2022-07-22
CN110951837B (en) 2022-08-26
CN110964787B (en) 2022-08-26
CN106434898B (en) 2020-02-21
CN110964786A (en) 2020-04-07
CN111020047A (en) 2020-04-17
CN106434884B (en) 2020-02-21
CN106434889A (en) 2017-02-22
CN106434895A (en) 2017-02-22
CN111020008B (en) 2022-10-28
CN110760570B (en) 2023-01-24
CN111020049A (en) 2020-04-17
CN106434883A (en) 2017-02-22
CN111020011A (en) 2020-04-17
CN110951837A (en) 2020-04-03
CN106434896B (en) 2020-02-18
CN111304348B (en) 2022-07-26
CN110964786B (en) 2022-08-26
CN106367492B (en) 2020-02-07
CN110964788A (en) 2020-04-07

Similar Documents

Publication Publication Date Title
CN106434899A (en) Method, primers and kit for quickly detecting bacillus cereus at constant temperature
CN106434890A (en) Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner
CN106367493A (en) Method for rapidly detecting salmonella at constant temperature, primer and applications of primer
CN106434900A (en) Method for conducting rapid constant-temperature detection on vibrio vulnificus and vibrio cholerae simultaneously, primer and kit
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
CN102534023A (en) RCA (rolling circle amplification) rapid detection primer and kit for Yersinia enterocolitica
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