CN106434900A - Method for conducting rapid constant-temperature detection on vibrio vulnificus and vibrio cholerae simultaneously, primer and kit - Google Patents

Method for conducting rapid constant-temperature detection on vibrio vulnificus and vibrio cholerae simultaneously, primer and kit Download PDF

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CN106434900A
CN106434900A CN201610780489.8A CN201610780489A CN106434900A CN 106434900 A CN106434900 A CN 106434900A CN 201610780489 A CN201610780489 A CN 201610780489A CN 106434900 A CN106434900 A CN 106434900A
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primer
vibrio
sequence
vibrio vulnificus
primer sets
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CN106434900B (en
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李雪玲
李园园
韦朝春
刘伟
贾犇
陆长德
李亦学
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Shanghai Institute of biomedical technology
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SHANGHAI CENTER FOR BIOINFORMATION TECHNOLOGY
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Abstract

The invention discloses a method for conducting rapid constant-temperature detection on vibrio vulnificus and vibrio cholerae simultaneously, a primer and a kit. The method comprises the steps that a genome DNA is extracted from a sample to be detected; by taking the genome DNA as a template and taking a primer group which can amplify a specific sequence shared by the vibrio vulnificus and the vibrio cholerae as the primer, a constant-temperature amplified reaction is conducted under an enzyme reaction system; by judging whether the reaction result is positive or not, whether the vibrio vulnificus and/or the vibrio cholerae exist/exists in the sample to be detected or not is determined. The detection method has high sensitivity and high specificity, the detection time is short, determination of results is easy, operation is convenient and rapid, the cost is low, and the method has a wide application prospect.

Description

Fast constant temperature detection Vibrio vulnificus and the method for comma bacillus, primer and examination simultaneously Agent box
Technical field
The invention belongs to biological technical field, be specifically related to a kind of detection Vibrio vulnificus of fast constant temperature simultaneously and comma bacillus Method, primer and kit.
Background technology
Vibrio vulnificus (Vibrio vulnificus) and comma bacillus (Vibrio cholerae) are often at marine food Two kinds of gram negative pathogenic bacterias of middle discovery, can cause the diseases such as primary septicemia, wound infection, acute gastroenteritis and cholera Disease.Therefore, for the prevention of both food-borne pathogens with detect extremely important.
Tradition Vibrio vulnificus and comma bacillus detection method are longer due to the detection cycle, operate relative complex, detection efficiency Relatively low, it is difficult to meet modern society for food-borne pathogens detection process high flux, high sensitivity, high specific, quick, just Prompt requirement.Recently as the development of nucleic acid molecules detection technique, researcher have also been developed the detection means such as PCR, but It is that the method needs special detecting instrument, therefore, be not appropriate for being widely used in especially enterprise of detection department of basic unit and produce The real-time on-site detection carrying out inside line.In order to ensure food security, be badly in need of quick, simple, accurately method detect food In Vibrio vulnificus and comma 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.
Have at present and separately design primer for two kinds of pathogenic bacteria (such as Vibrio vulnificus and comma bacillus), to both pathogenic bacteria The method carrying out LAMP detection respectively, but yet there are no the method using a set of LAMP primer simultaneously to detect two kinds of pathogenic bacteria And example.Therefore, a kind of method being capable of simultaneously fast constant temperature detection Vibrio vulnificus and comma bacillus is needed in industry badly, simultaneously full Detection department of foot basic unit, 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 present in existing LAMP technology design of primers a set of primer only Can the defect of a kind of pathogenic bacteria of detection, make full use of microbial genome sequence information abundant in current common data resource with And corresponding sequence analysis tools, it is designed for the specific primer sets simultaneously identifying Vibrio vulnificus and comma bacillus, and at this On the basis of formed high sensitivity, high specific detection kit.The present invention is based on the microbial genome in GenBank database Data resource (by data on August 5th, 2013) carries out Vibrio vulnificus and the design of comma bacillus LAMP primer, provides one The method of fast constant temperature augmentation detection Vibrio vulnificus and comma bacillus, primer sets and kit simultaneously.Use the detection of the present invention Method detects Vibrio vulnificus and comma bacillus simultaneously, has high sensitivity and high specific, and the detection time is short, and result judges letter Single, simple operation, the advantage of low cost.
Quickly detect the method for Vibrio vulnificus and vibrio cholera strain while the present invention proposes a kind of, described method include with Lower step：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that Vibrio vulnificus and cholera vibrio gene group public specificity alkali can be expanded The primer sets of basic sequence is primer, under enzyme reaction system, carries out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine whether testing sample exists Vibrio vulnificus and/or cholera Vibrios.
The method of the present invention Constant Temperature Detection Vibrio vulnificus and vibrio cholera strain simultaneously, extracts genome from testing sample DNA, which is template, with Vibrio vulnificus and comma bacillus specificity amplification primer group as primer, carries out isothermal amplification reactions, so After, by judging whether reaction result is positive, determine whether testing sample exists Vibrio vulnificus and/or comma bacillus.Its In, described enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the present invention, described Vibrio vulnificus and cholera vibrio gene group public specificity base sequence are No. GI and are Vibrio vulnficus gene group 2698869～2699116bp position of 320154846 and the cholera vibrio gene that No. GI is 147673035 Group 216343～216590bp bit sequence.
In the present invention, the described primer sets that can expand Vibrio vulnificus and cholera vibrio gene group public specificity base sequence For vibrio vulnficus gene group 2698869～2699116bp position that No. GI is 320154846 and the cholera that No. GI is 147673035 A part for vibrios genome 216343～216590bp position nucleotide sequence or a part for its complementary strand.Wherein, described wound Vibrios and cholera vibrio gene group public specificity base sequence refer to that only Vibrio vulnificus and cholera vibrio gene group are common And distinctive, and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand Vibrio vulnificus and cholera vibrio gene group specific base sequence includes but does not limits It in primer sets A, or is 68.4% and above primer with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence Group.
Primer sets A：
Upstream outer primer F3_A：5’-CGAGACTTGTGACGAGCTG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-TCCAAGCTGAGAAACGTCG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3’(SEQ ID NO：4).
In the present invention, described can expand Vibrio vulnificus and the primer sets of cholera vibrio gene group specific base sequence is all right Including be 68.4% and above primer sets with wall scroll sequence homology in aforementioned each primer sets sequence or its complementary strand sequence, should Primer sets includes but is not limited to following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-CGAGACTTGTGACGAGCTG-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-GCGCTATCCAAGCTGAGAA-3’(SEQ ID NO：6) (with primer B3_A5 '- TCCAAGCTGAGAAACGTCG-3 ' homology is 68.4%)；
Upstream inner primer FIP_B：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’(SEQ ID NO：7)；
Downstream inner primer BIP_B：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-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 positive, containing Vibrio vulnificus and/or cholera arc Bacterium；Band as stepped in electrophoretogram not expression characteristics, then testing sample is negative, and does not contains Vibrio vulnificus and comma bacillus.Institute Stating Turbidity measurement, being to detect by an unaided eye or transmissometer detection turbidity, detection pipe occurs substantially muddy, then testing sample is positive, Containing Vibrio vulnificus and/or comma bacillus；As muddy in having no, then testing sample is feminine gender, does not contains Vibrio vulnificus and comma bacillus. 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 positive, and contains Have Vibrio vulnificus and/or comma bacillus；Do not precipitate as at the bottom of reaction tube, then testing sample is negative, and does not contains Vibrio vulnificus and suddenly Random vibrios.
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 feminine gender, does not contains Vibrio vulnificus and comma bacillus；Such as reaction Rear color is green, then testing sample is the positive, containing Vibrio vulnificus and/or comma bacillus.When employing hydroxynaphthol blue conduct During developer, as after reaction, color is pansy, then testing sample is feminine gender, does not contains Vibrio vulnificus and comma bacillus；As instead Should color be sky blue afterwards, then testing sample be the positive, containing Vibrio vulnificus and/or comma bacillus.Described color developing detection, except Above by visually observing outside reaction result, it is also possible to carry out in real time or end point determination reaction result, passing through by detecting instrument Reasonably setting the threshold value of negative reaction, when the result of testing sample reaction is less than or equal to this threshold value, then testing sample is Feminine gender, does not contains Vibrio vulnificus and comma bacillus；When the result of testing sample reaction is more than this threshold value, then testing sample is sun Property, containing Vibrio vulnificus and/or comma bacillus.Described detecting instrument includes but is not limited to sepectrophotofluorometer, fluorescent quantitation 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 while Constant Temperature Detection Vibrio vulnificus and vibrio cholera strain method in primer.Institute Stating primer and including expanding the primer sets of Vibrio vulnificus and cholera vibrio gene group specific base sequence, it includes but is not limited to, The sequence of described primer be 2698869～2699116bp position of the vibrio vulnficus gene group that No. GI is 320154846 and No. GI be A part for the nucleotide sequence of 216343～216590bp position of the cholera vibrio gene group of 147673035 or the one of its complementary strand Part.
Wherein, described Vibrio vulnificus can be expanded and the primer sets of cholera vibrio gene group public specificity base sequence is selected from Any one group of following primer sets, or be selected from and wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence It is 68.4% and above arbitrary primer sets.Wherein, described primer sets includes but is not limited to following primer sets A.Described and aforementioned In primer sets sequence or its complementary strand sequence wall scroll sequence homology be 68.4% and above primer sets including but not limited to Lower primer sets B.
Primer sets A：
Upstream outer primer F3_A：5’-CGAGACTTGTGACGAGCTG-3’；
Downstream outer primer B3_A：5’-TCCAAGCTGAGAAACGTCG-3’；
Upstream inner primer FIP_A：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’；
Downstream inner primer BIP_A：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3’；
Primer sets B：
Upstream outer primer F3_B：5’-CGAGACTTGTGACGAGCTG-3’；
Downstream outer primer B3_B：5’-GCGCTATCCAAGCTGAGAA-3’；
Upstream inner primer FIP_B：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’；
Downstream inner primer BIP_B：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3’.
The present invention also provides a kind of for Constant Temperature Detection Vibrio vulnificus while above-mentioned with the examination in vibrio cholera strain method Agent box, it includes the described primer sets that can expand Vibrio vulnificus and cholera vibrio gene group specific base sequence.Reagent of the present invention In box, the described primer sets that can expand Vibrio vulnificus and cholera vibrio gene group public specificity base sequence, including but do not limit In being 147673035 genes with vibrio vulnficus gene group 2698869～2699116bp position that No. GI is 320154846 and No. GI A part for nucleotide sequence for group 216343～216590bp position or a part for its complementary strand are 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 68.4% 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 The including but not limited to complete genome DNA of Vibrio vulnificus and/or comma bacillus, portion gene group DNA, or comprise Vibrio vulnificus And/or the carrier of comma bacillus complete genome DNA or portion gene group DNA, two kinds of pathogenic bacteria gene groups DNA may be combined in one Rise, it is also possible to independently pack.
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 the total specific DNA sequence dna with Vibrio vulnificus and comma bacillus, therefore can be applicable to microorganism and divide The research fields such as class, comparative genomics, evolution, and the application such as microorganism detection.This carrier can be but not limit In plasmid vector (such as pBR322, pUC18, pUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda Deng) and artificial chromosome vectors (such as Bacterial artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, primer sets A is comprised Carrier pBR322-A, the carrier pBR322-B etc. comprising any one primer of primer sets B of any one primer.Comprise to draw Carrier bacteriophage lambda-the A of any one primer of thing group A, the carrier bacteriophage lambda-B comprising any one primer of primer sets B Deng.
The invention allows for the primer Constant Temperature Detection Vibrio vulnificus at the same time and suddenly selected from primer sets A, any one group of B Application in random vibrios.
The invention allows for the application in described kit Constant Temperature Detection Vibrio vulnificus at the same time and comma bacillus.
The invention allows for the application in described carrier Constant Temperature Detection Vibrio vulnificus at the same time and comma bacillus.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection Vibrio vulnificus simultaneously With the method for comma bacillus, primer/primer sets, detection reagent/kit, greater significance is had to the food security of China.This Invention beneficial effect includes：Invention wound vibrios and comma bacillus detection method is used to have primary first-order equation and can detect simultaneously Two kinds of pathogenic bacteria, high specificity, highly sensitive, the time is short in detection, result judges simple, simple operation, low cost and other advantages.With Conventional at present detection method is compared, and the constant-temperature amplification method that the present invention uses can be carried out under constant temperature, only need to use simple Thermostat, it is not necessary to the expensive instrument in PCR experiment, it is not necessary to carry out the steps such as electrophoresis detection to amplified production, thus, It is very suitable for being widely used in various circles of society and include that food safety detection department of basic unit promotes the use of, though special at molecular biology Also can fully apply in the environment of the relative deficiency of industry knowledge and skills basis.Can be by above-mentioned each optimum condition based on common sense in the field It is combined, all belong to scope.
Brief description
Fig. 1 shows the specific of the embodiment of the present invention 7 Vibrio vulnificus and comma bacillus Constant Temperature Detection method.
Fig. 2 shows the embodiment of the present invention 8 Vibrio vulnificus and the sensitivity of comma bacillus 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 Vibrio vulnificus and comma bacillus isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Vibrio vulnificus bacterial classification for detection derives from Chinese industrial Microbiological Culture Collection administrative center, numbering CICC10383 (=ATCC27562), comma bacillus bacterial classification derives from China General Microbiological DSMZ, numbering CGMCC 1.8676.Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract kit extraction genome DNA, DNA OD260/OD280Being 1.8～2.0, concentration is respectively 210.8ng/ μ L and 30.4ng/ μ L.
(2) with Vibrio vulnificus to be measured and cholera vibrio gene group DNA as template, the kit being respectively adopted autogamy (is shown in Table 1, table 2), and according to condition described in table 2, prepare reaction system, with Vibrio vulnificus and comma bacillus specificity amplification primer group For primer, carry out isothermal amplification reactions.Primer in embodiment 1～6 is respectively primer sets A, A, A, B, B, B.
(3) according to condition described in table 2, by electrophoresis detection, Turbidity measurement or color developing detection, amplification is carried out true Recognize.
As can be seen from Table 2, detection method and the primer sets being used and reaction system thereof can be right well Vibrio vulnificus and comma bacillus public specificity fragment carry out expanding and obtain testing result.Therefore, present invention could apply to Whether detection sample contains Vibrio vulnificus and/or comma bacillus.
Embodiment 7 Vibrio vulnificus and comma bacillus specific detection
Collect non-wound vibrios and comma bacillus 28 strain (1～25 in table 3 and Fig. 1,27～28 and 30), by these bacterial strains Cultivate respectively with Vibrio vulnificus and vibrio cholera strain (26 in table 3 and 29), take 1mL bacterium solution, use kit IA, carry Take DNA of bacteria, and with reference to the reaction system of embodiment 1 and condition, carry out LAMP amplification (primer sets is A) and adding respectively and develop the color Agent is observed.
Its testing result is as shown in table 3 and Fig. 1, and in Fig. 1,1～25 is respectively staphylococcus aureus, Staphylococcus aureus The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, gill fungus sample bacillus, listeria monocytogenes, Ying Nuoke Liszt Bacterium, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, paratyphoid B Salmonella Bacterium, shigella dysenteriae, Shigella bogdii, shigella flexneri, ETEC (gene containing Clostridium botulinum type A), cause Characteristic of disease ETEC, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic ETEC, Hemorrhagic ETEC, yersinia enterocolitica, artificial tuberculosis yersinia genus, 27～28 respectively secondary haemolysis arcs Bacterium and Freund vibrios, 30 is Song Shi Shigella, NTC：Negative control, P be 26 and No. 29 bacterial strain DNA mixed liquor (No. 26 and No. 29 are respectively Vibrio vulnificus and comma bacillus), i.e. the biased sample of Vibrio vulnificus and comma bacillus.In Fig. 1, only have and contain wound Product after the aggregate sample QC amplified reaction of vibrios and comma bacillus is rendered as bright green, is positive findings, such as P pipe institute Show.And the product after other non-wound vibrios and vibrio cholera strain and negative control amplified reaction is all rendered as orange, for the moon Property result, as the 1st～No. 25,27～No. 28 pipe and No. 30 pipe and NTC negative control pipe shown in.
By Fig. 1 and Biao 3 result it can be seen that detection kit of the present invention and detection method have good Vibrio vulnificus and Vibrio cholera strain is specific, i.e. only Vibrio vulnificus and the vibrio cholera strain amplification positive, other non-wound vibrios and cholera Cholerae strain is 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-wound vibrios and vibrio cholera strain and negative control amplified reaction is feminine gender knot Really, the product after Vibrio vulnificus and vibrio cholera strain amplified reaction is positive findings.
Additionally, specifically carry out theory analysis to primer sets A～B respectively, method is by the sequence between primers F 3 and B3 Carry out Bowtie comparison with the genome of the Vibrio vulnificus in GenBank database and/or comma bacillus, determine detection region, Then detection non-wound vibrios in common data base resource for the regional sequence and comma bacillus are carried out Blast comparison, detection Primer region and genome sequence matching degree.If matching degree is higher, then specifically poorer；If primer can not arrive in comparison simultaneously On non-detection species strain, it is specifically good to show.It was found that in the case that each bar primer at most allows three mispairing, respectively Primer sets has 6 primer comparisons to non-wound vibrios and comma bacillus at most simultaneously, is Freund vibrios (Vibrio Furnissii, GI=375129161), but have in its guiding region more mispairing (B3, F2, F1c are respectively arranged with two mispairing, F3, B2, B1c is respectively arranged with a mispairing), show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
Extract bacterium CICC10383's (Vibrio vulnificus) and CGMCC1.8676 (comma bacillus) as described in Example 2 DNA, uses kit IIB, and is separately added into reaction system according to 0.5fg, 5fg, 50fg and 500fg DNA ladder degree, and other are anti- Condition is answered to carry out LAMP amplification (primer sets is A) respectively with reference to the method for table 2 embodiment 2 and add developer to observe.Such as Fig. 2 institute Showing, 1-4 is respectively 0.5fg, 5fg, 50fg and 500fg, NTC：Negative control.Place with Vibrio vulnificus DNA as template in Fig. 2 Reason, the product that 0.5fg, 5fg, 50fg and 500fg are processed is rendered as bright green, is positive findings, the reaction of negative control Product is rendered as orange, is negative findings；Process with comma bacillus DNA as template, the reaction that 5fg, 50fg and 500fg are processed Product is rendered as bright green, is positive findings, and the product of 0.5fg and negative control is rendered as orange, is negative findings.Inspection Survey result to show, each reaction tube can be detected during minimum DNA containing 0.5fg～5fg (being equivalent to 1 bacterium), sensitivity Higher.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B, the DNA of as little as 5fg in each reaction tube Can be detected, detection sensitivity is higher.
Embodiment 8 versatility detects
Respectively theory analysis is carried out to the versatility of primer sets A～B, method be by the sequence between primers F 3 and B3 with The genome of the Vibrio vulnificus in GenBank database and/or comma bacillus carries out Bowtie comparison, determines detection region, so After will detection Vibrio vulnificus (3 genomes) in common data base resource for the regional sequence and comma bacillus (8 genomes) Carry out Blast comparison, the matching degree of detection primer region and genome sequence, if primer region is mated completely, then versatility Good.It was found that the primer region of each primer sets is mated completely with 11 genomes of above-mentioned Vibrio vulnificus and comma bacillus, table The versatility of bright each primer sets is all preferable.
The kit species of table 1 simultaneously Constant Temperature Detection Vibrio vulnificus and comma bacillus and mainly comprise composition
Reaction condition in the method for the table 2 embodiment 1-6 present invention simultaneously Constant Temperature Detection Vibrio vulnificus and comma bacillus and Testing result
Table 3 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.C) DNA profiling used by It is the mixed liquor (1 of 26 and No. 29 bacterial strain DNA：1).

Claims (15)

1. the method for simultaneously fast constant temperature detection Vibrio vulnificus and comma bacillus, 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 Vibrio vulnificus and cholera vibrio gene group public specificity base sequence can be expanded The primer sets of row, as primer, carries out isothermal amplification reactions under enzyme reaction system；
(3) by judging whether reaction result is positive, determine whether testing sample exists Vibrio vulnificus and/or cholera arc Bacterium；
Wherein, described Vibrio vulnificus and cholera vibrio gene group public specificity base sequence are the wound that No. GI is 320154846 Hinder 2698869～2699116bp position of vibrios genome and cholera vibrio gene group that No. GI is 147673035 216343～ 216590bp bit sequence.
2. the method for claim 1, it is characterised in that described Vibrio vulnificus and the cholera vibrio gene group of expanding has The primer sets sequence of specific base sequence is vibrio vulnficus gene group 2698869～2699116bp that No. GI is 320154846 The part of nucleotide sequence for position and cholera vibrio gene group 216343～216590bp position that No. GI is 147673035 or it is mutual Mend a part for chain.
3. method as claimed in claim 2, it is characterised in that described can expand Vibrio vulnificus and cholera vibrio gene group has The primer sets of specific base sequence is primer sets A；Or it is same with wall scroll sequence in described primer sets A sequence or its complementary strand sequence Source property is 68.4% and any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-CGAGACTTGTGACGAGCTG-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-TCCAAGCTGAGAAACGTCG-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’(SEQ ID NO：3)；
Downstream inner primer BIP_A：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-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 68.4% and above primer sets includes following primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-CGAGACTTGTGACGAGCTG-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-GCGCTATCCAAGCTGAGAA-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’(SEQ ID NO：7)；
Downstream inner primer BIP_B：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-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 Vibrio vulnificus simultaneously as claimed in claim 1 and comma bacillus method, its feature exists In described primer includes the primer sets that can expand Vibrio vulnificus and cholera vibrio gene group public specificity base sequence, its sequence It is classified as Vibrio vulnificus 2698869～2699116bp position that No. GI is 320154846 and the comma bacillus that No. GI is 147673035 A part for the nucleotide sequence of 216343～216590bp position of genome or a part for its complementary strand.
8. primer as claimed in claim 7, it is characterised in that described can expand Vibrio vulnificus and cholera vibrio gene group has The primer sets of specific base sequence is primer sets A；Or it is same with wall scroll sequence in described primer sets A sequence or its complementary strand sequence Source property is 68.4% and any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-CGAGACTTGTGACGAGCTG-3’；
Downstream outer primer B3_A：5’-TCCAAGCTGAGAAACGTCG-3’；
Upstream inner primer FIP_A：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’；Draw in downstream Thing BIP_A：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-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 68.4% and above primer sets includes primer sets B：
Primer sets B：
Upstream outer primer F3_B：5’-CGAGACTTGTGACGAGCTG-3’；
Downstream outer primer B3_B：5’-GCGCTATCCAAGCTGAGAA-3’；
Upstream inner primer FIP_B：5’-TGCAACTGCTGCATTCGCTGTATAAGGCCTTTAGTCGCCATG-3’；Draw in downstream Thing BIP_B：5’-CCAGCAGCGATTGCAGCAACACAATGCTAGCCGTCGTTC-3’.
10. the kit for Constant Temperature Detection Vibrio vulnificus and comma bacillus simultaneously, it is characterised in that described kit bag Include the primer as described in any one of claim 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 Vibrio vulnificus and comma bacillus simultaneously, it is characterised in that described kit Enzyme reaction system includes：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 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.
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 14. primers Constant Temperature Detection Vibrio vulnificus at the same time and comma bacillus, it is characterised in that described primer is for such as power Profit requires the primer described in any one of 7～9.
15. kits as described in any one of claim 10～12 or carrier as claimed in claim 13 constant temperature at the same time Application in detection Vibrio vulnificus and comma bacillus.
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