CN106367499A - Method for rapidly detecting vibrio vulnificus at constant temperature, primer and kit - Google Patents
Method for rapidly detecting vibrio vulnificus at constant temperature, primer and kit Download PDFInfo
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Abstract
The invention discloses a method for rapidly detecting vibrio vulnificus at constant temperature, a primer group and a kit. The method comprises the following steps: extracting genome DNA from a to-be-detected sample; taking the genome DNA as a template, taking a primer group capable of amplifying a specific sequence of vibrio vulnificus as a primer, and carrying out a constant temperature amplified reaction in an enzyme reaction system; determining whether vibrio vulnificus exists in the to-be-detected sample by judging whether the reaction result is positive. The detection method disclosed by the invention has the advantages of being high in sensitivity and specificity, short in detection time, simple in result determination, convenient and fast to operate, low in cost and wide in application prospects.
Description
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 320157827
2509～3725bp 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 2509～3725bp position of (No. gi is 320157827) or a part for its complementary strand.Wherein, institute
State vibrio vulnficus gene group-specific base sequence and refer to only specific to vibrio vulnficus gene group, and other microbial gene
Organize the base sequence not comprised.
Wherein, the described primer sets that can expand vibrio vulnficus gene group specific base sequence are including but not limited to selected from following
Any one group of each primer sets a～f, or selected from wall scroll sequence homology in this primer sets sequence or its complementary strand sequence being
50% and any one group of above primer sets.
Primer sets a:
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '
(seq id no:3)；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 ' (seq
Id no:4)；
Primer sets b:
Upstream outer primer f3_b:5 '-cgactagtgccaaatcac-3 ' (seq id no:5)；
Downstream outer primer b3_b:5 '-cgtgcgatcataataccg-3 ' (seq id no:6)；
Upstream inner primer fip_b:5 '-tctgattgaagggcatgtcaaatgatcatgctttcgaggt-3 ' (seq id
No:7)；
Downstream inner primer bip_b:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 ' (seq
Id no:8)；
Primer sets c:
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 ' (seq id no:9)；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 ' (seq id no:10)；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 ' (seq id
No:11)；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 ' (seq id
No:12)；
Primer sets d:
Upstream outer primer f3_d:5 '-tggtaccaagtcgaaatgt-3 ' (seq id no:13)；
Downstream outer primer b3_d:5 '-cctgatccagacttaccatt-3 ' (seq id no:14)；
Upstream inner primer fip_d:5 '-ggatgaatgtgagcaagttgggaaagatccattacgccg-3 ' (seq id
No:15)；
Downstream inner primer bip_d:5 '-accacaatgctgctttgctgtgaaaagtgaaccagact-3 ' (seq id
No:16)；
Primer sets e:
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 ' (seq id no:17)；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 ' (seq id no:18)；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 ' (seq
Id no:19)；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 ' (seq
Id no:20)；
Primer sets f:
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 ' (seq id no:21)；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 ' (seq id no:22)；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 ' (seq
Id no:23)；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 ' (seq
Id no:24).
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 50% and above primer sets, this primer sets include but
It is not limited to arbitrary primer sets of following primer sets g～l:
Primer sets g:
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 ' (seq id no:25) (with primer f3_a 5 '-
Gcagagaagatgatcgtttt-3 ' homology is 50%)；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 ' (seq id no:26)；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 ' (seq
Id no:27)；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 ' (seq
Id no:28)；
Primer sets h:
Upstream outer primer f3_h:5 '-ccaaatcacttgatcatgct-3 ' (seq id no:29) (with primer f3_b5 '-
Cgactagtgccaaatcac-3 ' homology is 50%)；
Downstream outer primer b3_h:5 '-cgtgcgatcataataccg-3 ' (seq id no:30)；
Upstream inner primer fip_h:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 ' (seq
Id no:31)；
Downstream inner primer bip_h:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 ' (seq
Id no:32)；
Primer sets i:
Upstream outer primer f3_i:5 '-ccaaatcacttgatcatgct-3 ' (seq id no:33)；
Downstream outer primer b3_i:5 '-tttctcaactacacgtcaag-3 ' (seq id no:34) is (mutual with primer f3_c
Mending chain 5 '-gcgttcgagtttctcaac-3 ' homology is 50%)；
Upstream inner primer fip_i:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 ' (seq
Id no:35)；
Downstream inner primer bip_i:5 '-ctctaagctcagtaccgccatagtgcgatcataataccgata-3 ' (seq
Id no:36)；
Primer sets j:
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 ' (seq id no:37)；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 ' (seq id no:38) (with primer b3_d 5 '-
Cctgatccagacttaccatt-3 ' homology is 50%)；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '
(seq id no:39)；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 ' (seq
Id no:40)；
Primer sets k:
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 ' (seq id no:41)；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 ' (seq id no:42) is (complementary with primer f3_e
Chain 5 '-caagttgctgatctccgcc-3 ' homology is 52.6%)；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaatgtttagccagatagaaagatcc-3 ' (seq
Id no:43)；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '
(seq id no:44)；
Primer sets l:
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 ' (seq id no:45)；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 ' (seq id no:46) is (with primer b3_f
5 '-tccaacacacaccattgagc-3 ' homology is 50%)；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 ' (seq
Id no:47)；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 ' (seq
Id no:48).
In the inventive method, the described primer sets that can expand vibrio vulnficus gene group-specific base sequence can comprise or
Do not comprise ring primer.Described ring primer can be one or more, including primer lf and/or lb.Described can expand Vibrio vulnificus
The primer sets of genome specificity base sequence are selected from following primer sets a ', c ', e ', f ', g ' and, j ', k ', any one group of l '；
Or be selected from and described primer sets a ', c ', e ', f ', g ', j ', k ' and, in l ' sequence or its complementary strand sequence, wall scroll sequence homology is
50% and any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Upper lantern primer lf_a:5 '-ttcgacttggtaccaagatcg-3 ' (seq id no:49)；
And/or, lower lantern primer lb_a:5 '-acattcatccaccacaatgct-3 ' (seq id no:50)；
Primer sets c ':
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Lower lantern primer lb_c:5 '-ctcaggcccaaacaccgagt-3 ' (seq id no:51)；
Primer sets e ':
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Upper lantern primer lf_e:5 '-agattgtcgaatggggtgacacc-3 ' (seq id no:52)；
Primer sets f ':
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '；
Lower lantern primer lb_f:5 '-ccggtacttctgcgtctgagga-3 ' (seq id no:53)；
Primer sets g ':
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Upper lantern primer lf_g:5 '-acaacatttcgacttggtacca-3 ' (seq id no:54)；
And/or, lower lantern primer lb_g:5 '-aaatgatttggcccaacttgc-3 ' (seq id no:55)；
Primer sets j ':
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Lower lantern primer lb_j:5 '-acattcatccaccacaatgct-3 ' (seq id no:56)；
Primer sets k ':
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaat-gtttagccagatagaaagatcc-3 '；Downstream
The upper lantern primer lf_k of inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 ':
5 '-aaacaatggactaaggcggc-3 ' (seq id no:57)；
And/or, lower lantern primer lb_k:5 '-tcacttttcaccagatcgcc-3 ' (seq id no:58)；
Primer sets l ':
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '
Lower lantern primer lb_l:5 '-ccggtacttctgcgtctgagga-3 ' (seq id no:59).
In a particular embodiment, for example, above-mentioned primer sets a ', in g ' and k ', can only comprise a upper lantern primer,
Or only comprise a lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.
In the inventive method, in specific embodiments (primer containing ring), 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,
Fip the and bip primer of 0.8-2.0 μm of ol/l, f3 the and b3 primer of 0.15-0.3 μm of ol/l, the lf of 0.4-1.0 μm of ol/l and/or
Lb primer, 0.16-0.64u/ μ l bst dna polymerase and 0-1.5mol/l glycine betaine.(do not contain in another specific embodiments
Ring primer) in, the enzyme reaction system of described constant-temperature amplification is: 1 × bst dna polymeric enzyme reaction buffer, 2-9mmol/l mg2+
(mgso4Or mgcl2), fip the and bip primer of 1.0-1.6mmol/l dntp, 0.8-2.0 μm of ol/l, 0.15-0.3 μm of ol/l's
F3 and b3 primer, 0.16-0.64u/ μ l bst dna polymerase and 0-1.5mol/l glycine betaine.Ring primer is favorably improved reaction
Efficiency.For example, 1 × bst dna polymeric enzyme reaction buffer can select 1 × thermopol reaction buffer, comprises
20mmol/l tris-hcl (ph8.8), 10mmol/l kcl, 10mmol/l (nh4)2So4,0.1%triton x-100,2mm
mgso4.Mgso in 1 × bst dna 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 2509～3725bp position for 320157827 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 50% 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 any one primer sets of following primer sets a～f.Described and front
State wall scroll sequence homology in primer sets sequence or its complementary strand sequence be 50% and above primer sets including but not limited to
Any one primer sets of lower primer sets g～l.
Primer sets a:
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:
5’-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3’；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Primer sets b:
Upstream outer primer f3_b:5 '-cgactagtgccaaatcac-3 '；
Downstream outer primer b3_b:5 '-cgtgcgatcataataccg-3 '；
Upstream inner primer fip_b:5 '-tctgattgaagggcatgtcaaatgatcatgctttcgaggt-3 '；
Downstream inner primer bip_b:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 '；
Primer sets c:
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Primer sets d:
Upstream outer primer f3_d:5 '-tggtaccaagtcgaaatgt-3 '；
Downstream outer primer b3_d:5 '-cctgatccagacttaccatt-3 '；
Upstream inner primer fip_d:5 '-ggatgaatgtgagcaagttgggaaagatccattacgccg-3 '；
Downstream inner primer bip_d:5 '-accacaatgctgctttgctgtgaaaagtgaaccagact-3 '；
Primer sets e:
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；Downstream
Inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Primer sets f:
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '；
Primer sets g:
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Primer sets h:
Upstream outer primer f3_h:5 '-ccaaatcacttgatcatgct-3 '；
Downstream outer primer b3_h:5 '-cgtgcgatcataataccg-3 '；
Upstream inner primer fip_h:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 '；
Downstream inner primer bip_h:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 '；
Primer sets i:
Upstream outer primer f3_i:5 '-ccaaatcacttgatcatgct-3 '；
Downstream outer primer b3_i:5 '-tttctcaactacacgtcaag-3 '；
Upstream inner primer fip_i:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 '；
Downstream inner primer bip_i:5 '-ctctaagctcagtaccgccatagtgcgatcataataccgata-3 '；
Primer sets j:
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Primer sets k:
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaatgtttagccagatagaaagatcc-3 '；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '；
Primer sets l:
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '.
The present invention is used in the primer in described Constant Temperature Detection Vibrio vulnificus method, described can expand vibrio vulnficus gene group
The primer sets of specific base sequence can also comprise or not comprise one or more ring primer；Described ring primer be lf and/or
lb.The described primer sets that can expand vibrio vulnficus gene group-specific base sequence are selected from following primer sets a ', c ', e ', f ',
Any one group of g ', j ', k ', l '；Or selected from and described primer sets a ', c ', e ', f ', g ', j ', k ', l ' sequence or its complementary strand
In sequence, wall scroll sequence homology is 50% and any one group of above primer sets:
Primer sets a ':
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:
5’-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3’；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Upper lantern primer lf_a:5 '-ttcgacttggtaccaagatcg-3 '；
And/or, lower lantern primer lb_a:5 '-acattcatccaccacaatgct-3 '；
Primer sets c ':
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Lower lantern primer lb_c:5 '-ctcaggcccaaacaccgagt-3 '；
Primer sets e ':
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Upper lantern primer lf_e:5 '-agattgtcgaatggggtgacacc-3 '；
Primer sets f ':
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '；
Lower lantern primer lb_f:5 '-ccggtacttctgcgtctgagga-3 '；
Primer sets g ':
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Upper lantern primer lf_g:5 '-acaacatttcgacttggtacca-3 '；
And/or, lower lantern primer lb_g:5 '-aaatgatttggcccaacttgc-3 '；
Primer sets j ':
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Lower lantern primer lb_j:5 '-acattcatccaccacaatgct-3 '；
Primer sets k ':
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaat-gtttagccagatagaaagatcc-3 '；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '
Upper lantern primer lf_k:5 '-aaacaatggactaaggcggc-3 '；
And/or, lower lantern primer lb_k:5 '-tcacttttcaccagatcgcc-3 '；
Primer sets l ':
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 ' downstream
Ring primer lb_l:5 '-ccggtacttctgcgtctgagga-3 '.
In a particular embodiment, above-mentioned primer sets a ', in g ' and k ', can only comprise a upper lantern primer, or only
Comprise a lower lantern primer, or comprise a upper lantern primer and a lower lantern primer simultaneously.In specific embodiments
In, described primer be respectively fip, bip, f3, b3, lf and lb shown in primer or with aforementioned primer sequence or its complementary strand sequence
Middle wall scroll primer homology is 50% and above primer.
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: 2509～3725bp 320157827)
A part for nucleotide sequence for position or a part for its complementary strand are as described primer sequence；Described primer includes but is not limited to institute
State any one primer sets of primer sets a, primer sets b, primer sets c, primer sets d, primer sets e, primer sets f etc..Also include but
Be not limited to using be 50% with wall scroll sequence homology in aforementioned primer sequence or its complementary strand sequence and above primer sets as
Primer；Including but not limited to primer sets g, primer sets h, primer sets i, primer sets j, primer sets k, primer sets l etc..
In test kit of the present invention, the described primer sets that can expand vibrio vulnficus gene group-specific base sequence can comprise
Or do not comprise one or more ring primer；Ring primer is as optional component.Described ring primer is lf and/or lb.Comprise ring primer
The primer sets of lf and/or lb include but is not limited to primer sets a ', c ', e ', f ', g ' and, j ', k ', l ' etc..In specific embodiments
In, lf the and/or lb ring primer of 0.4-1.0 μm of ol/l can be comprised in test kit of the present invention.In one embodiment, draw
The sequence of thing group be respectively fip, bip, f3, b3, lf and lb shown in primer or with foregoing sequences or its complementary strand sequence wall scroll
Primer homology is 50% and above primer.
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 selected from primer sets a～f, g～l, a ', c ', e ', f ',
Any one group of primer of g ', j ', k ', l '.This carrier, due to containing with Vibrio vulnificus specific dna sequence, therefore may be used
It is applied to the research fields such as microbial taxonomy, comparative genomics, evolution, and the application such as microorganism detection.This load
Body can be but not limited to plasmid vector (as pbr322, puc18, puc19, pbluescript m13, ti plasmid etc.), virus
Carrier (as bacteriophage lambda etc.) and artificial chromosome vectors (as Bacterial artificial chromosome bac, yeast artificial chromosome yac etc.).Example
As, the carrier pbr322-a of any one primer comprising primer sets a ... comprise the load of any one primer of primer sets g
Body pbr322-g ... comprise primer sets l ' the carrier pbr322-l ' of any one primer etc..Comprise primer sets a arbitrarily
Article one, the carrier bacteriophage lambda-a of primer ... the carrier bacteriophage lambda-g of any one primer comprising primer sets g ... comprise
Primer sets l ' the carrier bacteriophage lambda-l ' of any one primer etc..
The invention allows for being selected from primer sets a～f, g～l, a ', c ', e ', f ', g ', j ', k ', any one group of l '
Application in Constant Temperature Detection Vibrio vulnificus for the primer.
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 detection Vibrio vulnificus strain source in Chinese industrial Microbiological Culture Collection administrative center, number
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, c ', g ' (2 ring primer), g ' (1 ring primer), l, l '.
(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.Additionally, when being detected using detector, shortening reaction
Time is to also there being good Detection results (as embodiment 6) during 10min.Therefore, present invention could apply in detection sample being
No containing Vibrio vulnificus.
By above-described embodiment method, use primer sets b～f, primer sets g～k, primer sets a respectively ', e ', f ', j ' it is also possible to
Well Vibrio vulnificus specific fragment is expanded and obtained testing result.
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 respectively primer sets b～f, primer sets g～l, primer sets
A ', c ', e ', f ', g ', j ' and, k ', l ', by above-mentioned method for detecting specificity, respectively obtain same testing result, i.e. non-wound
Product after cholerae strain and negative control amplified reaction is negative findings, and the product after Vibrio vulnificus bacterial strain amplified reaction is sun
Property result.
Additionally, according to method described in table 1, respectively to primer sets a～f, primer sets g～l, primer sets a ', c ', e ', f ',
The specificity of g ', j ', k ', l ' carries out theory analysis, it was found that in the case that each bar primer at most allows three mispairing,
Each primer sets at most have two primers to compare on non-wound vibrio simultaneously, 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, 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, 10fg, ntc: negative control.The product that in Fig. 2,1ng, 100pg, 10pg, 1pg, 100fg are processed is rendered as viride nitens
Color, is positive findingses, and 10fg is processed and the product of negative control is rendered as orange, is negative findings.Testing result table
Bright, still can be detected during minimum dna containing 100fg (being approximately equivalent to 20 antibacterials) in each reaction tube, sensitivity is higher.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets b～f, primer sets g～l, primer sets respectively
A ', c ', e ', f ', g ', j ' and, k ', l ', in each reaction tube, as little as the dna of 1pg～100fg still can be detected, detection sensitivity
Higher.
Embodiment 9 versatility detects
According to method described in table 1, respectively to primer sets a～f, primer sets g～l, primer sets a ', c ', e ' and, f ', g ',
J ', k ', the versatility of l ' carries out theory analysis, it was found that No. 2 dyeing in the primer region of each primer sets and three plants of Vibrio vulnificus
The coupling completely of body (No. gi is respectively 320157827,326424156 and 37675660), can be used for above-mentioned three plants in theory
The detection of Vibrio vulnificus bacterial strain, shows that the versatility of each primer sets is all preferable.
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 (19)
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 320157827
2509～3725bp 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 No. gi be 320157827 the nucleotide sequence of vibrio vulnficus gene group 2509～3725bp position a part
Or a part for its complementary strand.
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 selected from any one group of following primer sets a～f；Or be selected from and described primer sets a～f sequence or its complementary strand sequence
Middle wall scroll sequence homology is 50% and any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 ' (seq id no:1)；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 ' (seq id no:2)；
Upstream inner primer fip_a:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 ' (seq
Id no:3)；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 ' (seq id
No:4)；
Primer sets b:
Upstream outer primer f3_b:5 '-cgactagtgccaaatcac-3 ' (seq id no:5)；
Downstream outer primer b3_b:5 '-cgtgcgatcataataccg-3 ' (seq id no:6)；
Upstream inner primer fip_b:5 '-tctgattgaagggcatgtcaaatgatcatgctttcgaggt-3 ' (seq id no:
7)；
Downstream inner primer bip_b:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 ' (seq id
No:8)；
Primer sets c:
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 ' (seq id no:9)；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 ' (seq id no:10)；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 ' (seq id no:
11)；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 ' (seq id no:
12)；
Primer sets d:
Upstream outer primer f3_d:5 '-tggtaccaagtcgaaatgt-3 ' (seq id no:13)；
Downstream outer primer b3_d:5 '-cctgatccagacttaccatt-3 ' (seq id no:14)；
Upstream inner primer fip_d:5 '-ggatgaatgtgagcaagttgggaaagatccattacgccg-3 ' (seq id no:
15)；
Downstream inner primer bip_d:5 '-accacaatgctgctttgctgtgaaaagtgaaccagact-3 ' (seq id no:
16)；
Primer sets e:
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 ' (seq id no:17)；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 ' (seq id no:18)；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 ' (seq id
No:19)；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 ' (seq id
No:20)；
Primer sets f:
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 ' (seq id no:21)；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 ' (seq id no:22)；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 ' (seq id
No:23)；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 ' (seq id
No:24).
4. method as claimed in claim 3 is it is characterised in that single with described primer sets a～f sequence or its complementary strand sequence
Bar sequence homology is 50% and above primer sets include any one group of following primer sets g～l:
Primer sets g:
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 ' (seq id no:25)；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 ' (seq id no:26)；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 ' (seq id
No:27)；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 ' (seq id
No:28)；
Primer sets h:
Upstream outer primer f3_h:5 '-ccaaatcacttgatcatgct-3 ' (seq id no:29)；
Downstream outer primer b3_h:5 '-cgtgcgatcataataccg-3 ' (seq id no:30)；
Upstream inner primer fip_h:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 ' (seq id
No:31)；
Downstream inner primer bip_h:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 ' (seq id
No:32)；
Primer sets i:
Upstream outer primer f3_i:5 '-ccaaatcacttgatcatgct-3 ' (seq id no:33)；
Downstream outer primer b3_i:5 '-tttctcaactacacgtcaag-3 ' (seq id no:34)；
Upstream inner primer fip_i:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 ' (seq id
No:35)；
Downstream inner primer bip_i:5 '-ctctaagctcagtaccgccatagtgcgatcataataccgata-3 ' (seq id
No:36)；
Primer sets j:
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 ' (seq id no:37)；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 ' (seq id no:38)；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 ' (seq
Id no:39)；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 ' (seq id
No:40)；
Primer sets k:
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 ' (seq id no:41)；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 ' (seq id no:42)；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaatgtttagccagatagaaagatcc-3 ' (seq id
No:43)；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 ' (seq id
No:44)；
Primer sets l:
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 ' (seq id no:45)；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 ' (seq id no:46)；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 ' (seq id
No:47)；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 ' (seq id
No:48).
5. method as claimed in claim 2 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence
Primer sets also comprise one or more ring primer；Described ring primer is lf and/or lb.
6. method as claimed in claim 5 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence
Primer sets be selected from following primer sets a ', c ', e ', f ', g ', j ', k ', any one group of l '；Or be selected from and described primer sets a ',
C ', e ', f ', g ', j ', k ', in l ' sequence or its complementary strand sequence wall scroll sequence homology be 50% and above primer sets it
Any one group:
Primer sets a ':
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:
5’-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3’；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Upper lantern primer lf_a:5 '-ttcgacttggtaccaagatcg-3 ' (seq id no:49)；
And/or, lower lantern primer lb_a:5 '-acattcatccaccacaatgct-3 ' (seq id no:50)；
Primer sets c ':
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Lower lantern primer lb_c:5 '-ctcaggcccaaacaccgagt-3 ' (seq id no:51)；
Primer sets e ':
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Upper lantern primer lf_e:5 '-agattgtcgaatggggtgacacc-3 ' (seq id no:52)；
Primer sets f ':
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '；
Lower lantern primer lb_f:5 '-ccggtacttctgcgtctgagga-3 ' (seq id no:53)；
Primer sets g ':
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Upper lantern primer lf_g:5 '-acaacatttcgacttggtacca-3 ' (seq id no:54)；
And/or, lower lantern primer lb_g:5 '-aaatgatttggcccaacttgc-3 ' (seq id no:55)；
Primer sets j ':
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Lower lantern primer lb_j:5 '-acattcatccaccacaatgct-3 ' (seq id no:56)；
Primer sets k ':
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaat-gtttagccagatagaaagatcc-3 '；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '
Upper lantern primer lf_k:5 '-aaacaatggactaaggcggc-3 ' (seq id no:57)；
And/or, lower lantern primer lb_k:5 '-tcacttttcaccagatcgcc-3 ' (seq id no:58)；
Primer sets l ':
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '
Lower lantern primer lb_l:5 '-ccggtacttctgcgtctgagga-3 ' (seq id no:59).
7. 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, including or do not include lf the and/or lb primer of 0.4-1.0 μm of ol/l.
8. 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.
9. 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 320157827
A part for the nucleotide sequence of 2509～3725bp position for genome or a part for its complementary strand.
10. primer as claimed in claim 9 is it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence
The primer sets of row are selected from any one group of following primer sets a～f；Or be selected from and described primer sets a～f sequence or its complementary strand sequence
In row, wall scroll sequence homology is 50% and any one group of above primer sets；
Primer sets a:
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:
5’-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3’；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Primer sets b:
Upstream outer primer f3_b:5 '-cgactagtgccaaatcac-3 '；
Downstream outer primer b3_b:5 '-cgtgcgatcataataccg-3 '；
Upstream inner primer fip_b:5 '-tctgattgaagggcatgtcaaatgatcatgctttcgaggt-3 '；
Downstream inner primer bip_b:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 '；
Primer sets c:
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Primer sets d:
Upstream outer primer f3_d:5 '-tggtaccaagtcgaaatgt-3 '；
Downstream outer primer b3_d:5 '-cctgatccagacttaccatt-3 '；
Upstream inner primer fip_d:5 '-ggatgaatgtgagcaagttgggaaagatccattacgccg-3 '；Downstream inner primer
Bip_d:5 '-accacaatgctgctttgctgtgaaaagtgaaccagact-3 '；
Primer sets e:
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Primer sets f:
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '.
11. primers as claimed in claim 10 it is characterised in that with described primer sets a～f sequence or its complementary strand sequence in
Wall scroll sequence homology is 50% and above primer sets include any one group of following primer sets g～l:
Primer sets g:
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Primer sets h:
Upstream outer primer f3_h:5 '-ccaaatcacttgatcatgct-3 '；
Downstream outer primer b3_h:5 '-cgtgcgatcataataccg-3 '；
Upstream inner primer fip_h:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 '；In downstream
Primer bip_h:5 '-gctgattacaaaactctaagctcagatatggccattcgcgatt-3 '；
Primer sets i:
Upstream outer primer f3_i:5 '-ccaaatcacttgatcatgct-3 '；
Downstream outer primer b3_i:5 '-tttctcaactacacgtcaag-3 '；
Upstream inner primer fip_i:5 '-gatcaacaaaactggctcactagctaacgcttccatcaatttg-3 '；
Downstream inner primer bip_i:5 '-ctctaagctcagtaccgccatagtgcgatcataataccgata-3 '；
Primer sets j:
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Primer sets k:
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaatgtttagccagatagaaagatcc-3 '；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '；
Primer sets l:
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '.
12. primers as claimed in claim 9 are it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence
The primer sets of row also comprise one or more ring primer；Described ring primer is lf and/or lb.
13. primers as claimed in claim 12 are it is characterised in that described can expand vibrio vulnficus gene group-specific base sequence
The primer sets of row are selected from following primer sets a ', c ', e ', f ', g ' and, j ', k ', any one group of l '；Or be selected from and described primer sets
In a ', c ', e ', f ', g ', j ', k ', l ' sequence or its complementary strand sequence, wall scroll sequence homology is 50% and above primer sets
Any one group:
Primer sets a ':
Upstream outer primer f3_a:5 '-gcagagaagatgatcgtttt-3 '；
Downstream outer primer b3_a:5 '-cttaccatttggcgatctg-3 '；
Upstream inner primer fip_a:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_a:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；Upper lantern is drawn
Thing lf_a:5 '-ttcgacttggtaccaagatcg-3 '；
And/or, lower lantern primer lb_a:5 '-acattcatccaccacaatgct-3 '；
Primer sets c ':
Upstream outer primer f3_c:5 '-gttgagaaactcgaacgc-3 '；
Downstream outer primer b3_c:5 '-ggtaccaagatcgaaagc-3 '；
Upstream inner primer fip_c:5 '-agcgtgttggctagatggattcaggattgagatagcgg-3 '；
Downstream inner primer bip_c:5 '-cttgctttgatcgtcgctgaggtgatacgctgaaaacg-3 '；
Lower lantern primer lb_c:5 '-ctcaggcccaaacaccgagt-3 '；
Primer sets e ':
Upstream outer primer f3_e:5 '-ggcggagatcagcaacttg-3 '；
Downstream outer primer b3_e:5 '-gaacccacatccatgttagacg-3 '；
Upstream inner primer fip_e:5 '-actacatcgcagaagagaccgcggattgctcaaaatggcgc-3 '；
Downstream inner primer bip_e:5 '-ggtagcgagatcgtgcgtgatgggggtattgaaccttatgcag-3 '；
Upper lantern primer lf_e:5 '-agattgtcgaatggggtgacacc-3 '；
Primer sets f ':
Upstream outer primer f3_f:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_f:5 '-tccaacacacaccattgagc-3 '；
Upstream inner primer fip_f:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_f:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '；
Lower lantern primer lb_f:5 '-ccggtacttctgcgtctgagga-3 '；
Primer sets g ':
Upstream outer primer f3_g:5 '-tgatcgttttcagcgtatc-3 '；
Downstream outer primer b3_g:5 '-gactattcattagatgccgac-3 '；
Upstream inner primer fip_g:5 '-cgtaatggatctttctatctggctaaccttgatcatgctttcg-3 '；
Downstream inner primer bip_g:5 '-ccgccttagtccattgttttaccattgtggtggatgaatgt-3 '；
Upper lantern primer lf_g:5 '-acaacatttcgacttggtacca-3 '；
And/or, lower lantern primer lb_g:5 '-aaatgatttggcccaacttgc-3 '；
Primer sets j ':
Upstream outer primer f3_j:5 '-cgcagagaagatgatcgt-3 '；
Downstream outer primer b3_j:5 '-acttaccatttggcgatct-3 '；
Upstream inner primer fip_j:5 '-ggatctttctatctggctaaacaacgtatcaccttgatcatgctt-3 '；
Downstream inner primer bip_j:5 '-aatgatttggcccaacttgctactattcattagatgccgac-3 '；
Lower lantern primer lb_j:5 '-acattcatccaccacaatgct-3 '；
Primer sets k ':
Upstream outer primer f3_k:5 '-cttgatcatgctttcgatct-3 '；
Downstream outer primer b3_k:5 '-ccttacaccaagttgctg-3 '；
Upstream inner primer fip_k:5 '-tgagcaagttgggccaaat-gtttagccagatagaaagatcc-3 '；
Downstream inner primer bip_k:5 '-gtcggcatctaatgaatagtctggcctgatccagacttaccatt-3 '
Upper lantern primer lf_k:5 '-aaacaatggactaaggcggc-3 '；
And/or, lower lantern primer lb_k:5 '-tcacttttcaccagatcgcc-3 '；
Primer sets l ':
Upstream outer primer f3_l:5 '-atgatttgcgaacccaccg-3 '；
Downstream outer primer b3_l:5 '-gctccctcaatccaacacac-3 '；
Upstream inner primer fip_l:5 '-gacgttaaccaagtttccccaccacgctcgggctaaattgaca-3 '；
Downstream inner primer bip_l:5 '-gcgccaataactcggccaatttgcatttgactcgccctctgaa-3 '
Lower lantern primer lb_l:5 '-ccggtacttctgcgtctgagga-3 '.
A kind of 14. test kits for Constant Temperature Detection Vibrio vulnificus are it is characterised in that described test kit includes such as claim 9
Primer described in～13 any one.
15. test kits as claimed in claim 14 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 16. 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, including or do not include 0.4-1.0 μm of ol/l lf and/or
Lb primer, 0.16-0.64u/ μ l bst dna polymerase, and the glycine betaine of 0-1.5mol/l.
A kind of 17. carriers are it is characterised in that described carrier comprises the primer as described in any one of claim 9～13.
Application in Constant Temperature Detection Vibrio vulnificus for 18. primers it is characterised in that described primer be as claim 9～13 it
Primer described in any one.
19. test kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are in Constant Temperature Detection
Application in Vibrio vulnificus.
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| CN202010018176.5A CN111004856B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method, primer group and kit for vibrio vulnificus |
| CN201610780407.XA CN106367499B (en) | 2016-08-30 | 2016-08-30 | Method, primer and kit for rapidly detecting vibrio vulnificus at constant temperature |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101348835A (en) * | 2008-09-09 | 2009-01-21 | 南开大学 | Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology |
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| 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 |
| US20160030531A1 (en) * | 2014-08-01 | 2016-02-04 | Northwestern University | Bacterial toxins and uses thereof as ras specific proteases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101348835A (en) * | 2008-09-09 | 2009-01-21 | 南开大学 | Reagent kit for detecting vibrio vulnificus by loop-mediated isothermal amplification technology |
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| PARK,J.H.等: "accession No. NC_014966.1：Vibrio vulnificus MO6-24/O chromosome II, complete sequence", 《GENBANK DATABASE》 * |
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| CN111004856A (en) | 2020-04-14 |
| CN106367499B (en) | 2020-02-21 |
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