CN106367500A - Method for rapidly detecting vibrio vulnificus at constant temperature, primer and application - Google Patents

Method for rapidly detecting vibrio vulnificus at constant temperature, primer and application Download PDF

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CN106367500A
CN106367500A CN201610780421.XA CN201610780421A CN106367500A CN 106367500 A CN106367500 A CN 106367500A CN 201610780421 A CN201610780421 A CN 201610780421A CN 106367500 A CN106367500 A CN 106367500A
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primer
sequence
primer sets
vibrio
vibrio vulnificus
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CN106367500B (en
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李雪玲
李园园
韦朝春
刘伟
贾犇
陆长德
李亦学
曹永梅
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Shanghai Wang Wang Food Group Co Ltd
Shanghai Industrial Institute For Research And Technology
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Shanghai Wang Wang Food Group Co Ltd
Shanghai Industrial Institute For Research And Technology
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    • C12Q1/6844—Nucleic acid amplification reactions
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Abstract

The invention discloses a method, primer set and kit for rapidly detecting vibrio vulnificus at constant temperature. The method includes the steps that genome DNA is extracted from a to-be-detected sample; the genome DNA serves as a template, the primer set capable of amplifying the specificity sequence of vibrio vulnificus serves as a primer, and a constant-temperature amplified reaction is carried out under an enzyme reaction system; whether vibrio vulnificus exists in the to-be-detected sample or not is determined by judging whether the reaction result is positive or not. The detection method has the high sensitivity and the high specificity, the detection time is short, the result judgment is simple, operating is convenient and fast, the cost is low, and broad application prospects are achieved.

Description

Fast constant temperature detects method, primer and the application of Vibrio vulnificus
Technical field
The invention belongs to biological technical field and in particular to a kind of fast constant temperature detect the method for Vibrio vulnificus, primer and Test kit.
Background technology
Vibrio vulnificus (vibrio vulnificus), also known as vibrio marinopraesens, are to find in sea water and some marine foods A kind of halophagia gram negative pathogenic bacteria.The mankind are it is usually because eat contaminated marine product raw, or Wound contact is subject to Pollution sea water or marine animal and infect this antibacterial, the diseases such as constitutional septicemia, traumatic infection and acute gastroenteritiss can be caused Shape, the mortality rate up to more than 50% of the septic shock wherein causing.In China, Vibrio vulnificus infection is multiple be born in coastal Area, is listed in one of eight high-risk greatly microorganisms in food pollution source.Additionally, had no bright by the initial symptom that Vibrio vulnificus cause Aobvious specificity, the therefore prevention to this bacterium and detection are particularly important.
At present, the detection for Vibrio vulnificus is mainly completed with biochemical identification by pathogen separation, but there is detection cycle Long, complex operation, it is difficult the shortcomings of differentiate close species.Recently as the development of nucleic acid molecules detection technique, with specificity Gene has been successfully applied to the laboratory diagnosiss of Vibrio vulnificus for the conventional pcr or real-time pcr technology that target is set up, tool There is the advantages of sensitivity is high, and detection time is short, but the method must be equipped with the instrument and equipment of costliness, need special operator. Therefore, it is not appropriate for the real-time on-site detection being widely used in carrying out inside basic unit's detection department especially enterprise's production line.For Guarantee food safety, be badly in need of quick, simple, accurate method to detect the Vibrio vulnificus in food.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, lamp) is in recent years Come a kind of novel constant-temperature nucleic acid amplification method to grow up, 4 specific primers are designed in 6 regions that this method is directed to target sequence (include 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), using a kind of dna polymerase with strand-displacement activity, it is incubated about 60min in constant temperature, you can complete core Sour 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 Do not need can complete under pcr instrument or fluorescent quantitation pcr instrument, constant temperature, naked eyes can determine whether reaction result, and sensitivity height, High specificity, response time is short, simple operation, low cost and other advantages.
Design of primers is a most key step in lamp technology, and Normal practice is by certain biological generally acknowledged spy to be detected Specific gene imports the online website (http://primerexplorer.jp/e) of lamp design of primers, sets relevant parameter life Become primer sets.That is, user must assure that the distinguished sequence that this target gene is species to be measured first.With patent of invention cn As a example 103160604 a and zl201310556940.4, they are respectively directed to the special base of the Vibrio vulnificus of document report Because of vvha gene and tolc gene order, Vibrio vulnificus detection is carried out using lamp technology.However, so-called " generally acknowledged spy Specific gene " is often based upon delayed knowledge, and is not based on ever-increasing microbial genome data and carries out necessary renewal, The primer obtaining based on this target-gene sequence is led to not necessarily to can ensure that its specificity and/or versatility in actual applications.This Invention table 1 illustrates the inadequate problem of primer versatility present in prior art.That is, institute in art methods The Vibrio vulnificus detection sequence using is actually and not all Vibrio vulnificus bacterial strain has, i.e. be possible to missing inspection Vibrio vulnificus Part bacterial strain.Similar problem exists in specific confirmation, i.e. be possible to for non-wound vibrio mistakenly to regard as wound Vibrio.Therefore, need a kind of Vibrio vulnificus detection method being able to ensure that specificity and versatility in industry badly, meet basic unit simultaneously Detection department, to quick, easily demand, easily can carry out real-time on-site detection 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 The not enough defect with specificity, makes full use of abundant microbial genome sequence information in current common data resource and phase The sequence analysis tools answered, are designed for the primer sets of specific recognition Vibrio vulnificus, and on this basis formed high sensitivity, High specific detection kit.The present invention is based on the microbial genome data resource in genbank data base (by 2013 August data on the 5th) carry out the design of Vibrio vulnificus lamp primer, there is provided a kind of side of fast constant temperature augmentation detection Vibrio vulnificus Method, primer sets and test kit.Detection method using the present invention detects Vibrio vulnificus, has high sensitivity and high specific, inspection The survey time is short, and result judgement is simple, simple operation, the advantage of low cost.
The present invention proposes a kind of method of quick detection Vibrio vulnificus bacterial strain, the method comprising the steps of:
(1) extract genome dna from testing sample；
(2) with described genome dna as template, so that the primer sets of vibrio vulnficus gene group-specific base sequence can be expanded For primer, under enzyme reaction system, carry out isothermal amplification reactions；
(3) pass through to judge whether reaction result is positive, determine and in testing sample, whether there is Vibrio vulnificus.
The method of Constant Temperature Detection Vibrio vulnificus bacterial strain of the present invention, extracts genome dna, with it as mould from testing sample Plate, with Vibrio vulnificus specificity amplification primer group as primer, carries out isothermal amplification reactions, then, by judging that reaction result is No for the positive, determine and in testing sample, whether there is Vibrio vulnificus.Wherein, described enzyme reaction system including but not limited to dna gathers Synthase reaction system.
In the present invention, described vibrio vulnficus gene group-specific base sequence is the Vibrio vulnificus that No. gi is 320154846 62063～62415bp bit sequence.
In the present invention, the described primer sets that can expand vibrio vulnficus gene group-specific base sequence are described genome The part of the nucleotide sequence of 62063～62415bp position of (No. gi is 320154846) or a part for its complementary strand.Wherein, Described vibrio vulnficus gene group-specific base sequence refers to only specific to vibrio vulnficus gene group, and other microorganism base Because organizing the base sequence not comprised.
Wherein, the described primer sets that can expand vibrio vulnficus gene group specific base sequence include but is not limited to primer sets a, Or selected from being 60% with wall scroll sequence homology in this primer sets sequence or its complementary strand sequence and above primer sets are arbitrarily One group.
Primer sets a:
Upstream outer primer f3_a:5 '-gtctccaaacttagaccaaa-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-tctactttgagcaaccgtat-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-gttgctttaatgttcgatatgggccaaaatttcgttatccagcg-3 ' (seq id no:3)；
Downstream inner primer bip_a:5 '-acattttcttttctagctcgcgtcagtacgaatcactacctt-3 ' (seq Id no:4).
In the present invention, the described primer sets that can expand vibrio vulnficus gene group specific base sequence can also include with aforementioned In each primer sets sequence or its complementary strand sequence, wall scroll sequence homology is 60% and above primer sets, this primer sets include but It is not limited to following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-gtactgaatacggttgctc-3 ' (seq id no:5) is (mutual with primer b3_a Mending chain 5 '-atacggttgctcaaagtaga-3 ' homology is 60%)；
Downstream outer primer b3_b:5 '-ctggatgaactcaggcta-3 ' (seq id no:6)；
Upstream inner primer fip_b:5 '-caacaatatcgcaccatgtggaagtagacaagtttgaagcc-3 ' (seq Id no:7)；
Downstream inner primer bip_b:5 '-tttcaaaggcatatcacgggtgtattactcaacgagttgcc-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, 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 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst dna polymerase and 0- 1.5mol/l glycine betaine.For example, 1 × bst dna polymeric enzyme reaction buffer 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 polymeric enzyme reaction buffer4With the magnesium ion mg in enzyme reaction system2+Do Merging treatment.
In the inventive method, the response procedures of described isothermal amplification reactions are 1. 60～65 DEG C incubation 10～90min, preferably Ground is 10～60min；2. 80 DEG C of terminating 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 agarose gel or polyacrylamide gel.Electrophoresis detection In result, band as stepped in electrophoretogram expression characteristicses, then testing sample is in that Vibrio vulnificus are positive, containing Vibrio vulnificus；As The electrophoretogram not stepped band of expression characteristicses, then testing sample is in that Vibrio vulnificus are negative.Described Turbidity measurement, is with the naked eye to see Examine or transmissometer detection turbidity, substantially muddiness in detection pipe, then testing sample is in that Vibrio vulnificus are positive, containing Vibrio vulnificus； As muddy in having no, then testing sample is that Vibrio vulnificus are negative.Whether can also there is precipitation after centrifugation in perusal reaction tube bottom, If there is precipitation at reaction tube bottom, testing sample is in that Vibrio vulnificus are positive, containing Vibrio vulnificus；As reaction tube bottom is not precipitated, then Testing sample is in that Vibrio vulnificus are 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).When using calcein or sybr green i work During for developer, after such as reacting, color is orange, then testing sample is that Vibrio vulnificus are negative；As after reaction, color is green, then Testing sample is that Vibrio vulnificus are positive, containing Vibrio vulnificus.When using hydroxynaphthol blue as developer, color after such as reacting For pansy, then testing sample is that Vibrio vulnificus are negative；As after reaction, color is sky blue, then testing sample is Vibrio vulnificus Positive.Described color developing detection, in addition to above by perusal reaction result it is also possible to by detecting instrument carry out in real time or End point determination reaction result, by the rational threshold value setting negative reaction, when the result of testing sample reaction is less than or equal to During this threshold value, then testing sample is that Vibrio vulnificus are negative；When the result of testing sample reaction is more than this threshold value, then testing sample Positive for Vibrio vulnificus.Described detecting instrument includes but is not limited to spectrofluorophotometer, fluorescent quantitation pcr instrument, constant-temperature amplification Micro-fluidic chip 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-in constant-temperature amplification Should add it is also possible to add it is therefore preferable to add before isothermal amplification reactions after isothermal amplification reactions complete before, permissible Effectively reduce the probability of 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 Vibrio vulnificus bacterial strain.Described primer includes expanding Increase the primer sets of vibrio vulnficus gene group specific base sequence, it includes but is not limited to, and the sequence of described primer for No. gi is A part for the nucleotide sequence of 62063～62415bp position for 320154846 vibrio vulnficus gene group or one of its complementary strand Point.
Wherein, the described primer sets that can expand vibrio vulnficus gene group-specific base sequence be selected from following primer sets it Any one group, or selected from being 60% and above with wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence Arbitrary primer sets.Wherein, described primer sets include but is not limited to following primer sets a.Described and aforementioned primer sets sequence or it is mutual Mending wall scroll sequence homology in chain-ordering is 60% and above primer sets including but not limited to following primer sets b.
Primer sets a:
Upstream outer primer f3_a:5 '-gtctccaaacttagaccaaa-3 '
Downstream outer primer b3_a:5 '-tctactttgagcaaccgtat-3 '
Upstream inner primer fip_a:
5’-gttgctttaatgttcgatatgggccaaaatttcgttatccagcg-3’；
Downstream inner primer bip_a:5 '-acattttcttttctagctcgcgtcagtacgaatcactacctt-3 '；
Primer sets b:
Upstream outer primer f3_b:5 '-gtactgaatacggttgctc-3 '；
Downstream outer primer b3_b:5 '-ctggatgaactcaggcta-3 '；
Upstream inner primer fip_b:5 '-caacaatatcgcaccatgtggaagtagacaagtttgaagcc-3 '；
Downstream inner primer bip_b:5 '-tttcaaaggcatatcacgggtgtattactcaacgagttgcc-3 '；
The present invention also provide a kind of for the test kit in above-mentioned Constant Temperature Detection Vibrio vulnificus bacterial strain method, it includes described The primer sets of vibrio vulnficus gene group specific base sequence can be expanded.In test kit of the present invention, described can expand Vibrio vulnificus base Because of the primer sets of group-specific base sequence, including but not limited to genome (No. gi: 62,063 320154846)～ A part for a part for the nucleotide sequence of 62415bp position or its complementary strand is as described primer sequence；Described primer include but It is not limited to described primer sets a.Also including but not limited to with wall scroll sequence homology in aforementioned primer sequence or its complementary strand sequence Property is 60% and above primer sets are as primer；Including but not limited to primer sets b.
In test kit of the present invention, also include bst dna polymerase buffer, bst dna polymerase, dntp solution, mg2+ (mgso4Or mgcl2One or more of) and glycine betaine.In one embodiment, test kit enzyme reaction system of the present invention Comprise 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+(mgso4Or mgcl2), 1.0-1.6mmol/l Fip the and bip primer of dntp, 0.8-2.0 μm of ol/l, f3 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst Dna polymerase and the glycine betaine of 0-1.5mol/l.For example, 1 × bst dna polymeric enzyme reaction buffer can from 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 polymeric enzyme reaction buffer4With enzyme reaction body Magnesium ion mg in system2+Do merging treatment.
In test kit of the present invention, also comprise positive control template.In one embodiment, described positive control template The including but not limited to full-length genome dna of Vibrio vulnificus, portion gene group dna, or comprise Vibrio vulnificus full-length genome dna or portion Divide the carrier of genome dna.
In test kit of the present invention, also comprise negative control template, described negative control template includes but is not limited to distilled water.
In test kit of the present invention, also comprise developer, developer includes but is not limited to calcein, sybr green i or Hydroxynaphthol blue.When developer is for calcein, in test kit, also comprise [mn2+], for example, mncl2.
In test kit of the present invention, also comprise distilled water.
In test 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 Due to containing with Vibrio vulnificus specific dna sequence, therefore can be applicable to microbial taxonomy, comparative genomics, Research field, and the application such as microorganism detection such as evolve.This carrier can be but not limited to plasmid vector (such as Pbr322, puc18, puc19, pbluescript m13, ti plasmid etc.), viral vector (as bacteriophage lambda etc.) and artificial coloring Body carrier (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, comprise carrier pbr322-b of any one primer of primer sets b etc..Comprise primer sets a arbitrarily Article one, the carrier bacteriophage lambda-a of primer, comprise carrier bacteriophage lambda-b of any one primer of primer sets b etc..
The invention allows for being selected from primer the answering in Constant Temperature Detection Vibrio vulnificus of any one group of primer sets a, b With.
The invention allows for application in Constant Temperature Detection Vibrio vulnificus for the described test kit.
The invention allows for application in Constant Temperature Detection Vibrio vulnificus for the described carrier.
The present invention provides a kind of side of simple and quick sensitive detection Vibrio vulnificus for technical field of food safety detection Method, primer/primer sets, detectable/test kit, the food safety to China has greater significance.Beneficial effect bag of the present invention Include: high specificity is had using invention wound vibrio detection method, sensitivity is high, detection time is short, result judgement is simple, behaviour Make convenient, low cost and other advantages.Compared with commonly using detection method at present, the constant-temperature amplification method that the present invention adopts, can be in constant temperature Under the conditions of carry out, only need to be using simple thermostat it is not necessary to expensive instrument in pcr experiment be it is not necessary to amplified production Carry out the steps such as electrophoresis detection, thus, it is very suitable for being widely used in various circles of society including food safety detection department of basic unit pushing away Wide use, even if also can fully apply in the environment of molecular biology Professional knowledge and skills base relative deficiency.Based on this Above-mentioned each optimum condition can be carried out combination in any by field general knowledge, all belong to the scope of the present invention.
Brief description
Fig. 1 shows the specificity of the embodiment of the present invention 7 Vibrio vulnificus Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 Vibrio vulnificus detection method.
Specific embodiment
In conjunction with specific examples below and accompanying drawing, the present invention is described in further detail, the protection content of the present invention It is not limited to following examples.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.The process of the enforcement present invention, Condition, reagent, experimental technique etc., in addition to the following content specially referring to, are universal knowledege and the common knowledge of this area, The present invention is not particularly limited content.
Embodiment 1-6 Vibrio vulnificus isothermal reaction system and detection method
Detected according to following (1)～(3) step:
(1) extraction of genome dna
For the Vibrio vulnificus strain source Chinese industrial Microbiological Culture Collection administrative center of detection, numbering Cicc10383 (=atcc27562).1ml bacterial culturess are taken to use the bacterial nucleic acid of Beijing Tiangeng bio-engineering corporation to extract Test kit extracts genome dna, dna od260/od280For 1.8, concentration is 210.8ng/ μ l.
(2) with vibrio vulnficus gene group dna to be measured as template, it is respectively adopted the test kit (being shown in Table 2, table 3) of autogamy, and press According to condition described in table 3, prepare reaction system, with Vibrio vulnificus specificity amplification primer group as primer, carry out constant-temperature amplification anti- Should.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, carry out amplification true Recognize.
As can be seen from Table 3, detection method and its primer sets being adopted and reaction system can be right well Vibrio vulnificus specific fragment is expanded and is obtained testing result.Therefore, present invention could apply in detection sample whether Containing Vibrio vulnificus.
Embodiment 7 Vibrio vulnificus specific detection
Collect 28 plants of non-wound vibrio (1～25 in table 4 and Fig. 1,27～29), by these bacterial strains and Vibrio vulnificus bacterial strain (in table 4 and Fig. 1 26) are cultivated respectively, take 1ml bacterium solution, using test kit ia, extract antibacterial dna, and with reference to embodiment 1 Reaction system and condition, carry out lamp amplification (primer sets be a) respectively and add developer observation.
As shown in table 4 and Fig. 1, in Fig. 1,1～25 is respectively staphylococcus aureuses, Staphylococcus aureus to its testing result The golden yellow subspecies of bacterium, staphylococcus epidermidiss, Rhodococcus equi, bacillus cereuss, gill fungus sample bacillus cereuss, monokaryon hypertrophy Listeria Bacterium, listeria innocua, Yi Shi listeria spp, intestinal Salmonella intestinal subspecies, Salmonella enteritidis, Salmonella typhimurium Bacterium, moscow' paratyphi B, shigella dysenteriae, Shigella bogdii, shigella flexneri, colon bacillus (contain Bacillus botulinuss a type gene), pathogenic colon bacillus, Diarrheogenil Escherichia coli, produce enterotoxin colon bacillus, intestinal Toxigenicity colon bacillus, hemorrhagic colon bacillus, the rugged Cronobacter sakazakii of slope, yersinia enterocolitica and vacation Tuberculosis yersinia, 27～29 are respectively vibrio parahaemolytious, Freund vibrio and vibrio cholera, ntc: negative control, and 26: wound Vibrio.In Fig. 1, only the product after Vibrio vulnificus bacterial strain amplified reaction is rendered as bright green, is positive findingses, such as No. 26 pipe Shown.And the product after other non-wound cholerae strain and negative control amplified reaction is all rendered as orange, it is negative findings, such as Shown in 1st～25,27～No. 29 pipes and ntc negative control pipe.
Detection kit of the present invention be can be seen that by Fig. 1 and Biao 4 result and detection method has good Vibrio vulnificus bacterium Strain specificity, i.e. only Vibrio vulnificus bacterial strain amplification is positive, other non-wound cholerae strain are feminine gender.
Prepare detection kit, the primer adopting in test kit is primer sets b, by above-mentioned method for detecting specificity, obtains Same testing result, i.e. the product after non-wound cholerae strain and negative control amplified reaction is negative findings, Vibrio vulnificus Product after bacterial strain amplified reaction is positive findingses.
Additionally, according to method described in table 1, respectively to primer sets a, the specificity of primer sets b carries out theory analysis, result Find, in the case that each bar primer at most allows three mispairing, each primer sets are up to a primer and compare non-wound arc On bacterium, show that the specificity of each primer sets is all preferable.
Embodiment 8 sensitivity technique
As described in Example 1 extract antibacterial cicc 10383 dna, using test kit ib, and according to 1ng, 100pg, 10pg, 1pg, 100fg and 10fg gradient adds reaction system, and other reaction conditions are carried out respectively with reference to the method for table 3 embodiment 1 Lamp amplification (primer sets are a) and addition developer are observed.As shown in Fig. 2 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, for sun Property result, 1pg, 100fg and 10fg are processed and the product of negative control is rendered as orange, are negative findings.Detection knot Fruit shows, minimum in each reaction tube (is approximately equivalent to 2 × 10 containing 10pg3Individual antibacterial) dna when still can be detected.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets b, as little as 100fg in each reaction tube Dna still can be detected, and detection sensitivity is higher.
Embodiment 9 versatility detects
According to method described in table 1, respectively to primer sets a, the versatility of primer sets b carries out theory analysis, it was found that (No. gi is respectively 320154846,326423644 Hes for the primer region of each primer sets and No. 1 chromosome of three plants of Vibrio vulnificus 37678184) coupling completely, can be used for the detection of above-mentioned three plants of Vibrio vulnificus bacterial strains in theory, shows the logical of each primer sets All preferable with property.
The versatility of primer and specificity analyses in the existing detection method of table 1 Vibrio vulnificus
Note: a) each Vibrio vulnificus bacterial strain has two chromosomes, by the sequence between primer f3 and b3 in patent and wound arc 6 DNA sequence sequences of 3 bacterial strains of bacterium carry out bowtie comparison, determine detection zone in gi 320154846#1/ Position in 320157827#2 genome, #1 represents the genome sequence of the item chromosome of this bacterial strain, and #2 represents this bacterial strain Article 2 chromosome genome sequence.B) detection zone sequence is carried out blast comparison in common data base resource, draw Object area mates good for versatility completely.C) detection zone sequence is carried out in common data base resource blast comparison, primer Region Matching degree is higher, and specificity is poorer；If primer can not compare in non-wound vibrio strain simultaneously, show that specificity is good.
The test kit species of table 2 Constant Temperature Detection Vibrio vulnificus and main constituents
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention Vibrio vulnificus and testing result
Table 4 test 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 findingses ,-: negative findings.

Claims (15)

1. a kind of fast constant temperature detects the method for Vibrio vulnificus it is characterised in that comprising the following steps:
(1) extract genome dna from testing sample；
(2) with described genome dna as template, using can expand the primer sets of vibrio vulnficus gene group-specific base sequence as Primer, carries out isothermal amplification reactions under enzyme reaction system；
(3) pass through to judge whether reaction result is positive, determine and in testing sample, whether there is Vibrio vulnificus；
Wherein, described vibrio vulnficus gene group-specific base sequence is the vibrio vulnficus gene group that No. gi is 320154846 62063～62415bp bit sequence.
2. the method for claim 1 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence Primer sets sequence be that No. gi is 320154846 one of the nucleotide sequence of vibrio vulnficus gene group 62063～62415bp position Point or its complementary strand a part.
3. method as claimed in claim 2 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence Primer sets be primer sets a；Or selected from wall scroll sequence homology in described primer sets a sequence or its complementary strand sequence be 60% And any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-gtctccaaacttagaccaaa-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-tctactttgagcaaccgtat-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-gttgctttaatgttcgatatgggccaaaatttcgttatccagcg-3 ' (seq id No:3)；
Downstream inner primer bip_a:5 '-acattttcttttctagctcgcgtcagtacgaatcactacctt-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 60% and above primer sets include following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-gtactgaatacggttgctc-3 ' (seq id no:5)；
Downstream outer primer b3_b:5 '-ctggatgaactcaggcta-3 ' (seq id no:6)；
Upstream inner primer fip_b:5 '-caacaatatcgcaccatgtggaagtagacaagtttgaagcc-3 ' (seq id No:7)；
Downstream inner primer bip_b:5 '-tttcaaaggcatatcacgggtgtattactcaacgagttgcc-3 ' (seq id No:8).
5. the method for claim 1 is it is characterised in that in step (2), described enzyme reaction system includes: 1 × bst Dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+, fip and bip of 1.0-1.6mmol/l dntp, 0.8-2.0 μm of ol/l Primer, f3 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst dna polymerase, the Radix Betae of 0-1.5mol/l Alkali.
6. the method for claim 1 is it is characterised in that the response procedures of described isothermal amplification reactions are: 1. 60～65 DEG C incubation 10～90min；2. 80 DEG C of terminating reaction 2～20min.
7. for the primer in Constant Temperature Detection Vibrio vulnificus method as claimed in claim 1 it is characterised in that described primer includes The primer sets of vibrio vulnficus gene group-specific base sequence can be expanded, its sequence is the Vibrio vulnificus that No. gi is 320154846 A part for the nucleotide sequence of 62063～62415bp position for genome or a part for its complementary strand.
8. primer as claimed in claim 7 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence Primer sets be primer sets a；Or selected from wall scroll sequence homology in described primer sets a sequence or its complementary strand sequence be 60% And any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-gtctccaaacttagaccaaa-3 '；
Downstream outer primer b3_a:5 '-tctactttgagcaaccgtat-3 '；
Upstream inner primer fip_a:
5’-gttgctttaatgttcgatatgggccaaaatttcgttatccagcg-3’；
Downstream inner primer bip_a:5 '-acattttcttttctagctcgcgtcagtacgaatcactacctt-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 60% and above primer sets include following primer sets b:
Primer sets b:
Upstream outer primer f3_b:5 '-gtactgaatacggttgctc-3 '；
Downstream outer primer b3_b:5 '-ctggatgaactcaggcta-3 '；
Upstream inner primer fip_b:5 '-caacaatatcgcaccatgtggaagtagacaagtttgaagcc-3 '；
Downstream inner primer bip_b:5 '-tttcaaaggcatatcacgggtgtattactcaacgagttgcc-3 '.
10. a kind of test kit for Constant Temperature Detection Vibrio vulnificus is it is characterised in that described test kit includes such as claim 7 Primer described in～9 any one.
11. test kits as claimed in claim 10 it is characterised in that its also include bst dna polymeric enzyme reaction buffer, Bst dna polymerase, dntp solution, mg2+, one or more of glycine betaine.
A kind of 12. test kits for Constant Temperature Detection Vibrio vulnificus are it is characterised in that the enzyme reaction system bag of described test kit Include: 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+, 1.0-1.6mmol/l dntp, 0.8-2.0 μm of ol/l Fip and bip primer, f3 the and b3 primer of 0.15-0.3 μm of ol/l, 0.16-0.64u/ μ l bst dna polymerase, and 0- The glycine betaine of 1.5mol/l.
A kind of 13. carriers are it is characterised in that described carrier comprises the primer as described in any one of claim 7～9.
Application in Constant Temperature Detection Vibrio vulnificus for 14. primers is it is characterised in that described primer is appointing as claim 7～9 Primer described in one.
15. test kits as described in any one of claim 10～12 or carrier as claimed in claim 13 are in Constant Temperature Detection Application in Vibrio vulnificus.
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