CN106434890A - Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner - Google Patents
Method, primers and kit for quickly detecting yersinia enterocolitica in constant-temperature manner Download PDFInfo
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Abstract
The invention discloses a method, a primer group and a kit for quickly detecting yersinia enterocolitica in a constant-temperature manner. The method comprises the following steps of extracting DNA (Deoxyribonucleic Acid) of a genome from a to-be-detected sample; using the DNA of the genome as a template, using a primer group capable of amplifying the specific sequence of the yersinia enterocolitica as primers, and carrying out constant-temperature amplification reaction under an enzyme reaction system; through judging whether a reaction result is positive or not, determining whether the yersinia enterocolitica exists in the to-be-detected sample or not. The detection method provided by the invention has high sensitivity and high specificity, is short in detection time, is simple in result decision, is convenient and quick to operate, is low in cost, and has a wide application prospect.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of fast constant temperature detection yersinia enterocolitica
Method, primer and kit.
Background technology
Yersinia enterocolitica (Yersinia enterocolitica) is distributed widely in nature, is can be
One of a few pathogenic entero becteria of growth under refrigerated storage temperature, in addition to causing gastrointestinal symptom, also can cause arthritis, tubercle
Property erythema and the illness such as mesenteric lymph is scorching, even cause septicemia, cause death.After this bacterium infects, clinical symptoms are usually failed to understand
Aobvious, easily cause mistaken diagnosis.Owing to this bacterium can survive at low ambient temperatures, therefore, freezer storage food is that modern society occurs to be somebody's turn to do
The important infection sources that bacterium infects, this bacterium has been classified as the conventional detection project of import and export food by many countries all.
Bacteria Identification in current food generally or utilizes tradition Physiology and biochemistry method to detect, due to the detection cycle
Longer, operate relative complex, detection efficiency is relatively low, it is difficult to meets modern society and detects process high pass for food-borne pathogens
Amount, high sensitivity, high specific, quick, require easily.Recently as the development of nucleic acid molecules detection technique, study people
Member have also been developed the detection means such as PCR and real-time fluorescence PCR, but both of which needs special detecting instrument, therefore,
It is not appropriate for the real-time on-site detection being widely used in carrying out inside detection department of basic unit especially enterprise's production line.For guaranteeing food
Product safety, be badly in need of quick, simple, accurately method detect the yersinia enterocolitica in food.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is in recent years
A kind of novel constant-temperature nucleic acid amplification method growing up, this method is for 4 specific primers of 6 region designs of target sequence
(including upstream and downstream outer primer F3 and B3 and upstream and downstream inner primer FIP and BIP, wherein FIP is made up of F1C and F2, and BIP is by B1C
With B2 composition), utilize a kind of archaeal dna polymerase with strand-displacement activity, be incubated about 60min at constant temperature, core can be completed
Acid amplified reaction, produces macroscopic byproduct of reaction-white magnesium pyrophosphate and precipitates (see document Notomi T, Okayama
H,Masubuchi H,Yonekawa T,Watanabe K,Amino N,Hase T.Loop-mediated isothermal
amplification of DNA,Nucleic Acids Research,2000Jun 15；28(12):E63).This technology has
Not needing PCR instrument or quantitative real time PCR Instrument, can completing under constant temperature, naked eyes i.e. can determine whether reaction result, and highly sensitive,
High specificity, reaction time is short, simple operation, low cost and other advantages.
Design of primers is a step the most key in LAMP technology, and Normal practice is by the spy generally acknowledging of certain biology to be detected
Specific gene imports the online website (http of LAMP primer design://primerexplorer.jp/e), set relevant parameter raw
Become primer sets.It is to say, user is it is first necessary to guarantee the distinguished sequence that this target gene is species to be measured.With patent of invention CN
As a example by 101182574B, for document report yersinia enterocolitica specific regions 16S-23S between district's sequence
Row, use LAMP technology to carry out yersinia enterocolitica detection.But, so-called " specific gene generally acknowledged " often base
It in delayed knowledge, and is not based on the renewal that ever-increasing microbial genome data carry out necessity, cause based on this target base
The primer obtaining because of sequence not necessarily can ensure that its versatility and/or specific in actual applications.Present invention table 1 illustrates
The problem that present in prior art, versatility cannot ensure.It is to say, the small intestine colon used in art methods
Scorching Yersinia ruckeri detection sequence actually not yersinia enterocolitica is common, i.e. be possible to missing inspection small intestine knot
The part bacterial strain of enteritis Yersinia ruckeri.Similar problem exists in specific confirmation, i.e. be possible to non-small intestine colon
Scorching Yersinia ruckeri regards as yersinia enterocolitica mistakenly.Therefore, need one in industry badly and be able to ensure that special
Property and the yersinia enterocolitica detection method of versatility, meet detection department of basic unit to quick, need easily simultaneously
Ask, real-time on-site detection can be carried out easily inside enterprise's production line.
Content of the invention
The technical problem to be solved in the present invention is to overcome primer versatility present in existing LAMP technology design of primers
With specific not enough defect, make full use of microbial genome sequence information abundant in current common data resource and phase
The sequence analysis tools answered, is designed for the primer sets of specific recognition yersinia enterocolitica, and on this basis
Form high sensitivity, high specific detection kit.The present invention provides based on the microbial genome data in GenBank database
Source (by data on August 5th, 2013) carries out the design of yersinia enterocolitica LAMP primer, provides a kind of quick
The method of constant-temperature amplification detection yersinia enterocolitica, primer sets and kit.Use the detection method inspection of the present invention
Surveying yersinia enterocolitica, having high sensitivity and high specific, the detection time is short, and result judges simple, and operation is just
Victory, the advantage of low cost.
The present invention proposes a kind of method of quick detection yersinia enterocolitica bacterial strain, and described method includes following
Step：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that yersinia enterocolitica genome specificity base can be expanded
The primer sets of sequence is primer, under enzyme reaction system, carries out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine whether testing sample exists enterocolitis yersinia genus
Salmonella.
The method of Constant Temperature Detection yersinia enterocolitica bacterial strain of the present invention, extracts genome from testing sample
DNA, which is template, with yersinia enterocolitica specificity amplification primer group as primer, carries out isothermal amplification reactions,
Then, by judging whether reaction result is positive, determine in testing sample whether there is yersinia enterocolitica.Its
In, described enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the present invention, described yersinia enterocolitica genome specificity base sequence is No. GI and is
169782～171532bp bit sequence of the yersinia enterocolitica of 123440403.
In the present invention, described can expand yersinia enterocolitica genome specificity base sequence primer sets be
A part for the nucleotide sequence of 169782～171532bp position of described genome (No. GI is 123440403) or its complementary strand
A part.Wherein, described yersinia enterocolitica genome specificity base sequence refers to only enterocolitis
Specific to Ademilson Salmonella genome, and the base sequence that other microbial genome do not comprise.
Wherein, the described primer sets that can expand yersinia enterocolitica genome specific base sequence include but not
It is limited to any one group selected from following primer sets A～I, or be selected from and wall scroll sequence in this primer sets sequence or its complementary strand sequence
Row homology is 50% and any one group of above primer sets.
Primer sets A：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’(SEQ
ID NO：3)；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’(SEQ
ID NO：4)；
Primer sets B：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’(SEQ
ID NO：7)；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’(SEQ
ID NO：8)；
Primer sets C：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’(SEQ ID NO：9)；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’(SEQ ID NO：10)；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’(SEQ ID
NO：11)；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’
(SEQ ID NO：12)；
Primer sets D：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’(SEQ ID NO：13)；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’(SEQ ID NO：14)；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’(SEQ ID
NO：15)；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’(SEQ ID
NO：16)；
Primer sets E：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：17)；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’(SEQ ID NO：18)；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’(SEQ ID
NO：19)；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’(SEQ ID
NO：20)；
Primer sets F：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’(SEQ ID NO：21)；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’(SEQ ID NO：22)；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’(SEQ
ID NO：23)；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’(SEQ
ID NO：24)；
Primer sets G：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’(SEQ ID NO：25)；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’(SEQ ID NO：26)；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’(SEQ ID
NO：27)；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’(SEQ
ID NO：28)；
Primer sets H：
Upstream outer primer F3_H：5’-CCATTGGGGGTAACAGTGTT-3’(SEQ ID NO：29)；
Downstream outer primer B3_H：5’-GCAGTGTGCCACCAATCA-3’(SEQ ID NO：30)；
Upstream inner primer FIP_H：5’-CAATCCCTGACAGAGCAGCCAGTTCCGGTCAGACAAACGAC-3’(SEQ
ID NO：31)；
Downstream inner primer BIP_H：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’(SEQ
ID NO：32)；
Primer sets I：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’(SEQ ID NO：33)；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’(SEQ ID NO：34)；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’(SEQ ID
NO：35)；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’(SEQ ID
NO：36).
In the present invention, the described primer sets that can expand yersinia enterocolitica genome specific base sequence also may be used
To include that with wall scroll sequence homology in aforementioned each primer sets sequence or its complementary strand sequence be 50% and above primer sets, should
Primer sets includes but is not limited to arbitrary primer sets of following primer sets J～R：
Primer sets J：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’(SEQ ID NO：37) (with primers F 3_A
5 '-TTTGTCTCAGTCAATTTCCC-3 ' homology is 50%)；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’(SEQ ID NO：38)；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’(SEQ ID
NO：39)；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’(SEQ ID
NO：40)；
Primer sets K：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’(SEQ ID NO：41)；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’(SEQ ID NO：42) (with primer B3_
B5 '-GTACTTACCCCTGCATTACGTG-3 ' homology is 81.8%)；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’(SEQ
ID NO：43)；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’(SEQ
ID NO：44)；
Primer sets L：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’(SEQ ID NO：45)；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’(SEQ ID NO：46) (with primer B3_C 5 '-
ACCAATACGGCTAACACC-3 ' homology is 77.8%)；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’(SEQ ID
NO：47)；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’
(SEQ ID NO：48)；
Primer sets M：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’(SEQ ID NO：49)；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’(SEQ ID NO：50) (with primer B3_D
5 '-CAAAAGGCATATCCCAGAA-3 ' homology is 52.6%)；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’
(SEQ ID NO：51)；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’(SEQ ID
NO：52)；
Primer sets N：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：53)；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’(SEQ ID NO：54) (with primer B3_E
5 '-AACATCCCAGCAAGAGTG-3 ' homology is 50%)；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’(SEQ
ID NO：55)；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’(SEQ
ID NO：56)；
Primer sets O：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：57)；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’(SEQ ID NO：58) (with primers F 3_F
Complementary strand 5 '-AAAGCTCATGCCTCGGACA-3 ' homology is 57.9%)；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’(SEQ ID
NO：59)；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’(SEQ ID
NO：60)；
Primer sets P：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’(SEQ ID NO：61)；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’(SEQ ID NO：62) (with primer B3_G 5 '-
TCGCATCCAACTCAACACC-3 ' homology is 52.6%)；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’(SEQ ID
NO：63)；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’(SEQ ID
NO：64)；
Primer sets Q：
Upstream outer primer F3_Q：5’-TAACAGTGTTTCAGCGGCTC-3’(SEQ ID NO：65) (with primers F 3_H
5 '-CCATTGGGGGTAACAGTGTT-3 ' homology is 50%)；
Downstream outer primer B3_Q：5’-GCAGTGTGCCACCAATCA-3’(SEQ ID NO：66)；
Upstream inner primer FIP_Q：5’-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3’(SEQ ID
NO：67)；
Downstream inner primer BIP_Q：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’(SEQ
ID NO：68)；
Primer sets R：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’(SEQ ID NO：69)；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’(SEQ ID NO：70) (with primer B3_I 5 '-
CGTTTGGTTGCAAAACAG-3 ' homology is 50%)；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’(SEQ ID
NO：71)；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’(SEQ ID
NO：72).
In the inventive method, the described primer that can expand yersinia enterocolitica genome specificity base sequence
Group can be including but not limited to a ring primer.Preferably, described ring primer can be one, including ring primer LF or LB.Institute
State the primer sets that can expand yersinia enterocolitica genome specificity base sequence selected from following primer sets A ', B ',
C ', D ', E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', P ', R ' any one group；Or be selected from and described primer sets A ', B ',
C ', D ', E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', P ', R ' wall scroll sequence homology is in sequence or its complementary strand sequence
50% and any one group of above primer sets：
Primer sets A '：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Lower lantern primer LB_A：5’-CAGTTATTGCATTTACTGGTGTGC-3’(SEQ ID NO：73)；
Primer sets B '：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_B：5’-CGGCACTGGGGTTGTTTATTGAGT-3’(SEQ ID NO：74)；
Primer sets C '：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_C：5’-TTCTGGCTACCAACCGGAAT-3’(SEQ ID NO：75)；
Primer sets D '：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Upper lantern primer LF_D：5’-GGCTAACACCACAATGGCTTTA-3’(SEQ ID NO：76)；
Primer sets E '：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Upper lantern primer LF_E：5’-TTTGCCAATAGCACACCAGTA-3’(SEQ ID NO：77)；
Primer sets F '：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Upper lantern primer LF_F：5’-CCAGGCATAATTTGCCAACGTGC-3’(SEQ ID NO：78)；
Primer sets G '：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_G：5’-ACCCCCAATGGCGTAAACGTT-3’(SEQ ID NO：79)；
Primer sets I '：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_I：5’-TGGGCTGCGTTATTCATAGG-3’(SEQ ID NO：80)；
Primer sets J '：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Upper lantern primer LF_J：5’-GCATTATCAGTCAGTAAGTCACAC-3’(SEQ ID NO：81)；
Primer sets K '：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_K：5’-CGGCACTGGGGTTGTTTATTGAGT-3’(SEQ ID NO：82)；
Primer sets L '：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_L：5’-TTCTGGCTACCAACCGGAAT-3’(SEQ ID NO：83)；
Primer sets M '：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；Lower lantern
Primer LB_M：5’-CATATCTTCCGGAGGCGAAA-3’(SEQ ID NO：84)；
Primer sets N '：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；Under
Lantern primer LB_N：5’-CATTGTTTTAGTGATGGGGGC-3’(SEQ ID NO：85)；
Primer sets O '：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Lower lantern primer LB_O：5’-TGGATTATTGATTCCCTGAAACTCC-3’(SEQ ID NO：86)；
Primer sets P '：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_P：5’-ACCCCCAATGGCGTAAACGTT-3’(SEQ ID NO：87)；
Primer sets R '：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_R：5’-TGGGCTGCGTTATTCATAGG-3’(SEQ ID NO：88).
In the inventive method, in a specific embodiments (primer containing ring), the enzyme reaction system of described constant-temperature amplification is：
1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L dNTP,
FIP and the BIP primer of 0.8-2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, LF or LB of 0.4-1.0 μm of ol/L
Primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.(do not contain ring in another specific embodiments
Primer) in, the enzyme reaction system of described constant-temperature amplification is：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+
(MgSO4Or MgCl2), FIP and the BIP primer of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L, 0.15-0.3 μm of ol/L's
F3 and B3 primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase and 0-1.5mol/L glycine betaine.Ring primer is favorably improved reaction
Efficiency.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 × Thermopol reaction buffer, comprises
20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,2mM
MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With the magnesium ion Mg in enzyme reaction system2+Do merging treatment.
In the inventive method, the response procedures of described isothermal amplification reactions hatches 10～90min, preferably for 1. 60～65 DEG C
Ground is 10～60min；2. 80 DEG C terminate reaction 2～20min.The present invention does not limit and realizes this by other suitable response procedures
Invention detection method.
In the inventive method, detection method includes but is not limited to electrophoresis detection, Turbidity measurement or color developing detection etc..Described electricity
Swimming detection, preferably gel electrophoresis assays, can be Ago-Gel, it is also possible to be polyacrylamide gel.Electrophoresis detection
In result, band as stepped in electrophoretogram expression characteristics, then testing sample is that yersinia enterocolitica is positive, contains
Yersinia enterocolitica；Band as stepped in electrophoretogram not expression characteristics, then testing sample is enterocolitis
Ademilson Salmonella is negative.Described Turbidity measurement, is to detect by an unaided eye or transmissometer detection turbidity, and detection pipe occurs substantially muddy, then
Testing sample is that yersinia enterocolitica is positive, containing yersinia enterocolitica；As muddy in having no, then to be measured
Sample is that yersinia enterocolitica is negative.Also can visually observe whether have precipitation at the bottom of reaction tube after centrifugal, if instead
Should have precipitation at the bottom of pipe, then testing sample is that yersinia enterocolitica is positive, containing yersinia enterocolitica；As
Do not precipitate at the bottom of reaction tube, then testing sample is that yersinia enterocolitica is negative.
Described color developing detection, is addition developer, including but not limited to calcein (50 μM) or SYBR in reaction tube
Green I (30-50 ×), or hydroxynaphthol blue (i.e. HNB, 120-150 μM).Make when using calcein or SYBR Green I
During for developer, as after reaction, color is orange, then testing sample is that yersinia enterocolitica is negative；Such as face after reaction
Look is green, then testing sample is that yersinia enterocolitica is positive, containing yersinia enterocolitica.Work as employing
When hydroxynaphthol blue is as developer, as after reaction, color is pansy, then testing sample is Yersinia enterocolitica
Bacterium is negative；As after reaction, color is sky blue, then testing sample is that yersinia enterocolitica is positive.Described colour developing inspection
Survey, in addition to above by visually observing reaction result, it is also possible to carried out in real time by detecting instrument or end point determination reaction is tied
Really, by the rational threshold value setting negative reaction, when the result of testing sample reaction is less than or equal to this threshold value, then to be measured
Sample is that yersinia enterocolitica is negative；When the result of testing sample reaction is more than this threshold value, then testing sample is
Yersinia enterocolitica is positive.Described detecting instrument includes but is not limited to sepectrophotofluorometer, quantitative fluorescent PCR
Instrument, constant-temperature amplification micro-fluidic chip foranalysis of nucleic acids instrument and Genie II isothermal duplication fluorescence detecting system etc..
In described color developing detection, according to calcein or hydroxynaphthol blue as developer, can be anti-at constant-temperature amplification
Added before should, it is also possible to add after isothermal amplification reactions completes, it is therefore preferable to add before isothermal amplification reactions, permissible
The effective possibility reducing reaction pollution.According to SYBR Green I as developer, then complete in isothermal amplification reactions
Add afterwards.According to calcein as developer, then while adding 50 μM of calceins in enzyme reaction system, add
0.6-1mM[Mn2+], for example, the MnCl of 0.6-1mM2.
Present invention also offers for the primer in the method for Constant Temperature Detection yersinia enterocolitica bacterial strain.Described
Primer includes the primer sets that can expand yersinia enterocolitica genome specific base sequence, and it includes but is not limited to,
The sequence of described primer is 169782～171532bp of the yersinia enterocolitica genome that No. GI is 123440403
A part for the nucleotide sequence of position or a part for its complementary strand.
Wherein, the described primer sets that can expand yersinia enterocolitica genome specificity base sequence selected from
Under any one group of each primer sets, or selected from wall scroll sequence homology in described each primer sets sequence or its complementary strand sequence being
50% and above arbitrary primer sets.Wherein, described primer sets includes but is not limited to any one of following primer sets A～I and draws
Thing group.Described is 50% and above primer sets bag with wall scroll sequence homology in aforementioned primer sets sequence or its complementary strand sequence
Include but be not limited to any one primer sets of following primer sets J～R.
Primer sets A：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Primer sets B：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Primer sets C：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Primer sets D：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Primer sets E：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Primer sets F：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Primer sets G：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Primer sets H：
Upstream outer primer F3_H：5’-CCATTGGGGGTAACAGTGTT-3’；
Downstream outer primer B3_H：5’-GCAGTGTGCCACCAATCA-3’；
Upstream inner primer FIP_H：5’-CAATCCCTGACAGAGCAGCCAGTTCCGGTCAGACAAACGAC-3’；Downstream
Inner primer BIP_H：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’；
Primer sets I：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Primer sets J：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Primer sets K：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Primer sets L：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Primer sets M：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；
Primer sets N：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；
Primer sets O：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Primer sets P：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Primer sets Q：
Upstream outer primer F3_Q：5’-TAACAGTGTTTCAGCGGCTC-3’；
Downstream outer primer B3_Q：5’-GCAGTGTGCCACCAATCA-3’；
Upstream inner primer FIP_Q：5’-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3’；
Downstream inner primer BIP_Q：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’；
Primer sets R：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’.
The present invention is in the primer in described Constant Temperature Detection yersinia enterocolitica method, and described energy amplification is little
The primer sets of Yersinia enterocolitica genome specificity base sequence can also be including but not limited to a ring primer；Excellent
Selection of land, described ring primer is one, including LF or LB.Described can expand yersinia enterocolitica genome specificity alkali
The primer sets of basic sequence is selected from following primer sets A ', B ', C ', D ', E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', P ', R ' it
Any one group；Or be selected from and described primer sets A ', B ', C ', D ', E ', F ', G ' and, I ', J ', K ', L ' and, M ', N ', O ', P ' and, R ' sequence
In row 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’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Lower lantern primer LB_A：5’-CAGTTATTGCATTTACTGGTGTGC-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_B：5’-CGGCACTGGGGTTGTTTATTGAGT-3’；
Primer sets C '：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_C：5’-TTCTGGCTACCAACCGGAAT-3’；
Primer sets D '：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Upper lantern primer LF_D：5’-GGCTAACACCACAATGGCTTTA-3’；
Primer sets E '：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Upper lantern primer LF_E：5’-TTTGCCAATAGCACACCAGTA-3’；
Primer sets F '：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Upper lantern primer LF_F：5’-CCAGGCATAATTTGCCAACGTGC-3’；
Primer sets G '：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_G：5’-ACCCCCAATGGCGTAAACGTT-3’；
Primer sets I '：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_I：5’-TGGGCTGCGTTATTCATAGG-3’；
Primer sets J '：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Upper lantern primer LF_J：5’-GCATTATCAGTCAGTAAGTCACAC-3’；
Primer sets K '：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_K：5’-CGGCACTGGGGTTGTTTATTGAGT-3’；
Primer sets L '：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_L：5’-TTCTGGCTACCAACCGGAAT-3’；
Primer sets M '：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；
Lower lantern primer LB_M：5’-CATATCTTCCGGAGGCGAAA-3’；
Primer sets N '：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；
Lower lantern primer LB_N：5’-CATTGTTTTAGTGATGGGGGC-3’；
Primer sets O '：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Lower lantern primer LB_O：5’-TGGATTATTGATTCCCTGAAACTCC-3’；
Primer sets P '：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_P：5’-ACCCCCAATGGCGTAAACGTT-3’；
Primer sets R '：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_R：5’-TGGGCTGCGTTATTCATAGG-3’.
The present invention also provides a kind of for the reagent in above-mentioned Constant Temperature Detection yersinia enterocolitica bacterial strain method
Box, it includes the described primer sets that can expand yersinia enterocolitica genome specific base sequence.Reagent of the present invention
In box, the described primer sets that can expand yersinia enterocolitica genome specificity base sequence, including but not limited to
With genome (No. GI:123440403) part for the nucleotide sequence of 169782～171532bp position or the one of its complementary strand
Part is as described primer sequence；Described primer include but is not limited to described primer sets A, primer sets B ..., primer sets I appoint
One primer sets of meaning etc..Also include but is not limited to wall scroll sequence homology in aforementioned primer sequence or its complementary strand sequence to be
50% and above primer sets as primer；Including but not limited to primer sets J, primer sets K ..., primer sets R etc..
In kit of the present invention, described can expand drawing of yersinia enterocolitica genome specificity base sequence
Thing group can be including but not limited to a ring primer；Ring primer is as optional component.Preferably, described ring primer is one, bag
Include LF or LB.The primer sets comprising ring primer LF or LB includes but is not limited to primer sets A ', B ', C ', D ', E ' and, F ', G ', I ',
J ', K ', L ', M ', N ', O ' and, P ', R ' etc..In a particular embodiment, kit of the present invention can comprise 0.4-1.0 μm of ol/
LF or the LB ring primer of L.In one embodiment, the sequence of primer sets be respectively FIP, BIP, F3, B3, LF or FIP,
Primer shown in BIP, F3, B3, LB or be 50% and above with foregoing sequences or its complementary strand sequence wall scroll primer homology
Primer.
In kit of the present invention, also include Bst DNA polymerase buffer liquid, Bst archaeal dna polymerase, dNTP solution, Mg2+
(MgSO4Or MgCl2) and glycine betaine in one or more.In one embodiment, kit enzyme reaction system of the present invention
Comprise 1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+(MgSO4Or MgCl2), 1.0-1.6mmol/L
FIP and the BIP primer of dNTP, 0.8-2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst
Archaeal dna polymerase and the glycine betaine of 0-1.5mol/L.For example, 1 × Bst DNA polymerase reaction buffer solution can select 1 ×
Thermopol reaction buffer, comprises 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4,0.1%Triton X-100,2mM MgSO4.MgSO in 1 × Bst DNA polymerase reaction buffer solution4With enzyme reaction body
Magnesium ion Mg in system2+Do merging treatment.
In kit of the present invention, also comprise positive control template.In one embodiment, described positive control template
Include but is not limited to complete genome DNA, portion gene group DNA of yersinia enterocolitica, or comprise enterocolitis
Yersinia ruckeri complete genome DNA or the carrier of portion gene group DNA.
In kit of the present invention, also comprising negative control template, described negative control template includes but is not limited to distilled water.
In kit of the present invention, also comprising developer, developer includes but is not limited to calcein, SYBR Green I or
Hydroxynaphthol blue.When developer is calcein, kit also comprises [Mn2+], for example, MnCl2.
In kit of the present invention, also comprise distilled water.
In kit of the present invention, also comprise nucleic acid extraction reagent.
The invention allows for a kind of carrier, described carrier comprises selected from primer sets A～I, J～R, A ', B ', C ', D ',
Any one group of primer of E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', P ', R '.This carrier has small intestine knot owing to containing
The specific DNA sequence dna of enteritis Yersinia ruckeri, therefore can be applicable to microbial taxonomy, comparative genomics, evolution etc. and grinds
Study carefully the applications such as field, and microorganism detection.This carrier can be but not limited to plasmid vector (as pBR322, pUC18,
PUC19, pBluescript M13, Ti-plasmids etc.), viral vectors (such as bacteriophage lambda etc.) and artificial chromosome vectors be (such as bacterium
Artificial chromosome BAC, yeast artificial chromosome YAC etc.).For example, the carrier of any one primer of primer sets A is comprised
PBR322-A, the carrier pBR322-J ... of any one the primer comprising primer sets J comprise primer sets A ' any one draw
The carrier pBR322-A ' of thing.Carrier bacteriophage lambda-the A of any one the primer comprising primer sets A, to comprise primer sets J arbitrarily
Article one, the carrier bacteriophage lambda-J ... of primer comprises primer sets A ' the carrier bacteriophage lambda-A ' etc. of any one primer.
The invention allows for selected from primer sets A～I, J～R, A ', B ', C ', D ', E ', F ', G ' and, I ', J ', K ', L ',
M ', N ', O ', P ', application in Constant Temperature Detection yersinia enterocolitica for the primer of any one group of R '.
The invention allows for application in Constant Temperature Detection yersinia enterocolitica for the described kit.
The invention allows for application in Constant Temperature Detection yersinia enterocolitica for the described carrier.
The present invention is that technical field of food safety detection provides a kind of simple and quick sensitive detection enterocolitis
The method of Ademilson Salmonella, primer/primer sets, detection reagent/kit, have greater significance to the food security of China.This
Bright beneficial effect includes：Yersinia enterocolitica detection method of the present invention is used to have high specificity, highly sensitive, inspection
The survey time is short, result judges simple, simple operation, low cost and other advantages.Compared with conventional at present detection method, the present invention uses
Constant-temperature amplification method, can carry out under constant temperature, only need to use simple thermostat, it is not necessary to high in PCR experiment
Your instrument, it is not necessary to carry out the steps such as electrophoresis detection to amplified production, thus, it is very suitable for being widely used in various circles of society and include
Food safety detection department of basic unit promotes the use of, even if at the environment of molecular biology professional knowledge and skills base relative deficiency
Under also can fully apply.Above-mentioned each optimum condition can be combined based on common sense in the field, all belong to the present invention and protect model
Enclose.
Brief description
Fig. 1 shows the specific of the embodiment of the present invention 7 yersinia enterocolitica Constant Temperature Detection method.
Fig. 2 shows the sensitivity of the embodiment of the present invention 8 yersinia enterocolitica detection method.
Detailed description of the invention
Being combined to lower specific embodiments and the drawings, the present invention is described in further detail, the protection content of the present invention
It is not limited to following example.Under the spirit and scope without departing substantially from inventive concept, those skilled in the art it is conceivable that change
Change and advantage is all included in the present invention, and with appending claims as protection domain.Implement the present invention process,
Condition, reagent, experimental technique etc., outside the lower content mentioned specially, be universal knowledege and the common knowledge of this area,
The present invention is not particularly limited content.
Embodiment 1-6 yersinia enterocolitica isothermal reaction system and detection method
Detect according to following (1)～(3) step：
(1) extraction of genomic DNA
Yersinia enterocolitica bacterial classification for detection derives from the management of Chinese industrial Microbiological Culture Collection
The heart, numbering CICC21565.Taking 1mL bacterial cultures uses the bacterial nucleic acid of Beijing Tian Gen bio-engineering corporation to extract kit
Extract genomic DNA, DNA OD260/OD280Being 1.8, concentration is 364ng/ μ L.
(2) with yersinia enterocolitica genomic DNA to be measured as template, be respectively adopted autogamy kit (see
Table 2, table 3), and according to condition described in table 3, prepare reaction system, draw with yersinia enterocolitica specific amplification
Thing group is primer, carries out isothermal amplification reactions.Primer in embodiment 1～6 is respectively primer sets A, A ', I, N, P ', R '.
(3) according to condition described in table 3, by electrophoresis detection, Turbidity measurement or color developing detection, amplification is carried out true
Recognize.
As can be seen from Table 3, detection method and the primer sets being used and reaction system thereof can be right well
Yersinia enterocolitica specific fragment carries out expanding and obtains testing result.Additionally, use detector to carry out when working as
During detection, shorten the reaction time to also there being good Detection results (such as embodiment 6) during 10min.Therefore, the present invention can apply
Whether contain yersinia enterocolitica in detection sample.
By above-described embodiment method, use primer sets B～H, primer sets J～M, primer sets O～R, primer sets B respectively ', C ',
D ', E ', F ', G ', I ', J ' and, K ', L ', M ', N ' and, O ' also can be well to yersinia enterocolitica specific fragment
Carry out expanding and obtain testing result.
Embodiment 7 yersinia enterocolitica specific detection
Collect non-yersinia enterocolitica 28 strain (in table 4 and Fig. 1 1～23,25～29), by these bacterial strains with
Yersinia enterocolitica bacterial strain (in table 4 and Fig. 1 24) is cultivated respectively, takes 1mL bacterium solution, uses kit IA,
Extract DNA of bacteria, and with reference to the reaction system of embodiment 1 and condition, carry out LAMP amplification (primer sets is A) and adding respectively and show
Toner is observed.
Its testing result is as shown in table 4 and Fig. 1, and in Fig. 1,1～23 is respectively staphylococcus aureus, Staphylococcus aureus
The golden yellow subspecies of bacterium, MRSE, Rhodococcus equi, bacillus cereus, gill fungus sample bacillus, listeria monocytogenes,
Ying Nuoke Listeria, listeria ivanovii, intestines salmonella intestines subspecies, Bacterium enteritidis, salmonella typhimurium, B-mode
Salmonella paratyphi, shigella dysenteriae, Shigella bogdii, shigella flexneri, ETEC (contain clostridium botulinum
A type gene), pathogenic ETEC, Diarrheogenil Escherichia coli, product enterotoxin ETEC, enterotoxigenic big
Intestines Escherichia, hemorrhagic ETEC and the rugged Cronobacter sakazakii of slope, 25～29 are respectively artificial tuberculosis yersinia genus, wound
Hinder vibrios, vibrio parahaemolytious, Freund vibrios and yersinia enterocolitica, NTC：Negative control, 24：Enterocolitis
Ademilson Salmonella.In Fig. 1, only the product after yersinia enterocolitica bacterial strain amplified reaction is rendered as bright green, for sun
Property result, as No. 24 pipe shown in.And after other non-yersinia enterocolitica bacterial strains and negative control amplified reaction
Product is all rendered as orange, is negative findings, as the 1st～No. 23,25～No. 29 is managed and shown in NTC negative control pipe.
By Fig. 1 and Biao 4 result it can be seen that detection kit of the present invention and detection method have good enterocolitis
Pestis strain is specific, i.e. the only yersinia enterocolitica bacterial strain amplification positive, other non-enterocolitises
Pestis strain is feminine gender.
Preparation detection kit, the primer using in kit is respectively primer sets B～I, primer sets J～R, primer sets
A ', B ', C ', D ', E ', F ' and, G ', I ', J ', K ' and, L ', M ', N ', O ' and, P ', R ', by above-mentioned method for detecting specificity, respectively obtain
Same testing result, i.e. the product after non-yersinia enterocolitica bacterial strain and negative control amplified reaction is feminine gender
As a result, the product after yersinia enterocolitica bacterial strain amplified reaction is positive findings.
Additionally, according to method described in table 1, respectively to primer sets A～I, primer sets J～R, primer sets A ', B ', C ', D ',
E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', P ', R ' specifically carry out theory analysis, it was found that at each bar primer
In the case of three mispairing of many permissions, each primer sets has two primer comparisons to non-yersinia enterocolitica at most simultaneously
On, show the specific all preferable of each primer sets.
Embodiment 8 sensitivity technique
As described in Example 2 extract bacterium CICC 21565 DNA, use kit IB, and according to 50ng, 5ng,
500pg, 50pg, 5pg, 500fg, 50fg and 5fg DNA ladder degree adds reaction system, and other reaction conditions are with reference to table 3 embodiment 1
Method carry out respectively LAMP amplification (primer sets is A) and add developer observe.As in figure 2 it is shown, 1-8 be respectively 50ng,
5ng, 500pg, 50pg, 5pg, 500fg, 50fg and 5fg, NTC：Negative control.50ng, 5ng, 500pg, 50pg, 5pg in Fig. 2
The product processing with 500fg is rendered as bright green, is positive findings, the product of 50fg, 5fg process and negative control
It is rendered as orange, be negative findings.Testing result shows, minimum containing 500fg (being approximately equivalent to 100 bacteriums) in each reaction tube
DNA when still can be detected, sensitivity is higher.
By above-mentioned detection method, other Step By Conditions ibid, use primer sets B～I, primer sets J～R, primer sets respectively
A ', B ', C ', D ', E ', F ' and, G ', I ', J ', K ' and, L ', M ', N ', O ' and, P ', R ', as little as 5pg～500fg 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～I, primer sets J～R, primer sets A ', B ', C ' and, D ', E ',
F ', G ', I ', J ', K ', L ' and, M ', N ', O ', P ', the versatility of R ' carries out theory analysis, it was found that the primer of each primer sets
Region is complete with three strain yersinia enterocoliticas (No. GI is respectively 123440403,332159624 and 386307442)
Full coupling, may be used for the detection of above-mentioned three strain yersinia enterocolitica bacterial strains in theory, shows the logical of each primer sets
All preferable by property.
The versatility of primer and specifically analysis in the existing detection method of table 1 yersinia enterocolitica
Note：A) three genome (GI by the sequence between primers F in patent 3 and B3 and yersinia enterocolitica
It number is respectively the 123440403rd, 332159624 and 386307442) carry out Bowtie comparison, determine detection region at No. GI
Position in 123440403 genomes.B) detection regional sequence is carried out in common data base resource Blast comparison, primer
It is good that region is mated completely for versatility.C) detection regional sequence is carried out in common data base resource Blast comparison, guiding region
Territory matching degree is higher, specifically poorer；If primer can not simultaneously comparison in non-yersinia enterocolitica strain, show
Specifically good.
The kit species of table 2 Constant Temperature Detection yersinia enterocolitica and mainly comprise composition
Reaction condition in the method for table 3 embodiment 1-6 Constant Temperature Detection of the present invention yersinia enterocolitica and inspection
Survey result
Table 4 tests bacterial strain uses therefor and testing result
Note：a)CGMCC：China General Microbiological DSMZ, CICC：Chinese industrial Microbiological Culture Collection manages
Center, CMCC：Chinese medicine bacteria culture preservation administrative center.b)+：Positive findings ,-：Negative findings.
Claims (19)
1. the method for a fast constant temperature detection yersinia enterocolitica, it is characterised in that comprise the following steps：
(1) from testing sample, genomic DNA is extracted；
(2) with described genomic DNA as template, so that yersinia enterocolitica genome specificity base sequence can be expanded
Primer sets as primer, under enzyme reaction system, carry out isothermal amplification reactions；
(3) by judging whether reaction result is positive, determine in testing sample whether there is yersinia enterocolitica；
Wherein, described yersinia enterocolitica genome specificity base sequence is the small intestine that No. GI is 123440403
169782～171532bp bit sequence of colitis Yersinia ruckeri genome.
2. the method for claim 1, it is characterised in that the described yersinia enterocolitica genome that can expand is special
The primer sets sequence of opposite sex base sequence be yersinia enterocolitica genome that No. GI is 123,440,403 169782～
A part for the nucleotide sequence of 171532bp position or a part for its complementary strand.
3. method as claimed in claim 2, it is characterised in that described to expand yersinia enterocolitica genome special
The primer sets of opposite sex base sequence is selected from any one group of following primer sets A～I；Or selected from described primer sets A～I sequence or
In its complementary strand sequence, wall scroll sequence homology is 50% and any one group of above primer sets；
Primer sets A：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’(SEQ ID NO：1)；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’(SEQ ID NO：2)；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’(SEQ ID
NO：3)；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’(SEQ ID
NO：4)；
Primer sets B：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’(SEQ ID NO：5)；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’(SEQ ID NO：6)；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’(SEQ ID
NO：7)；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’(SEQ ID
NO：8)；
Primer sets C：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’(SEQ ID NO：9)；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’(SEQ ID NO：10)；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’(SEQ ID NO：
11)；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’(SEQ
ID NO：12)；
Primer sets D：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’(SEQ ID NO：13)；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’(SEQ ID NO：14)；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’(SEQ ID NO：
15)；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’(SEQ ID NO：
16)；
Primer sets E：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：17)；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’(SEQ ID NO：18)；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’(SEQ ID NO：
19)；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’(SEQ ID NO：
20)；
Primer sets F：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’(SEQ ID NO：21)；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’(SEQ ID NO：22)；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’(SEQ ID
NO：23)；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’(SEQ ID
NO：24)；
Primer sets G：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’(SEQ ID NO：25)；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’(SEQ ID NO：26)；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’(SEQ ID NO：
27)；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’(SEQ ID
NO：28)；
Primer sets H：
Upstream outer primer F3_H：5’-CCATTGGGGGTAACAGTGTT-3’(SEQ ID NO：29)；
Downstream outer primer B3_H：5’-GCAGTGTGCCACCAATCA-3’(SEQ ID NO：30)；
Upstream inner primer FIP_H：5’-CAATCCCTGACAGAGCAGCCAGTTCCGGTCAGACAAACGAC-3’(SEQ ID
NO：31)；
Downstream inner primer BIP_H：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’(SEQ ID
NO：32)；
Primer sets I：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’(SEQ ID NO：33)；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’(SEQ ID NO：34)；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’(SEQ ID NO：
35)；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’(SEQ ID NO：
36).
4. method as claimed in claim 3, it is characterised in that single with described primer sets A～I sequence or its complementary strand sequence
Bar sequence homology is 50% and above primer sets includes any one group of following primer sets J～R：
Primer sets J：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’(SEQ ID NO：37)；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’(SEQ ID NO：38)；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’(SEQ ID NO：
39)；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’(SEQ ID NO：
40)；
Primer sets K：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’(SEQ ID NO：41)；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’(SEQ ID NO：42)；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’(SEQ ID
NO：43)；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’(SEQ ID
NO：44)；
Primer sets L：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’(SEQ ID NO：45)；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’(SEQ ID NO：46)；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’(SEQ ID NO：
47)；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’(SEQ
ID NO：48)；
Primer sets M：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’(SEQ ID NO：49)；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’(SEQ ID NO：50)；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’(SEQ
ID NO：51)；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’(SEQ ID NO：
52)；
Primer sets N：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：53)；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’(SEQ ID NO：54)；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’(SEQ ID
NO：55)；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’(SEQ ID
NO：56)；
Primer sets O：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’(SEQ ID NO：57)；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’(SEQ ID NO：58)；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’(SEQ ID NO：
59)；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’(SEQ ID NO：
60)；
Primer sets P：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’(SEQ ID NO：61)；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’(SEQ ID NO：62)；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’(SEQ ID NO：
63)；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’(SEQ ID NO：
64)；
Primer sets Q：
Upstream outer primer F3_Q：5’-TAACAGTGTTTCAGCGGCTC-3’(SEQ ID NO：65)；
Downstream outer primer B3_Q：5’-GCAGTGTGCCACCAATCA-3’(SEQ ID NO：66)；
Upstream inner primer FIP_Q：5’-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3’(SEQ ID NO：
67)；
Downstream inner primer BIP_Q：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’(SEQ ID
NO：68)；
Primer sets R：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’(SEQ ID NO：69)；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’(SEQ ID NO：70)；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’(SEQ ID NO：
71)；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’(SEQ ID NO：
72).
5. method as claimed in claim 2, it is characterised in that described to expand yersinia enterocolitica genome special
The primer sets of opposite sex base sequence also comprises a ring primer；Described ring primer is LF or LB.
6. method as claimed in claim 5, it is characterised in that described to expand yersinia enterocolitica genome special
The primer sets of opposite sex base sequence is selected from following primer sets A ', B ', C ', D ', E ' and, F ', G ', I ', J ' and, K ', L ', M ', N ', O ',
P ', R ' any one group；Or be selected from and described primer sets A ', B ', C ', D ', E ', F ', G ' and, I ', J ', K ', L ' and, M ', N ', O ',
P ', R ' wall scroll sequence homology is 50% and any one group of above primer sets in sequence or its complementary strand sequence：
Primer sets A '：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Lower lantern primer LB_A：5’-CAGTTATTGCATTTACTGGTGTGC-3’(SEQ ID NO：73)；
Primer sets B '：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_B：5’-CGGCACTGGGGTTGTTTATTGAGT-3’(SEQ ID NO：74)；
Primer sets C '：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_C：5’-TTCTGGCTACCAACCGGAAT-3’(SEQ ID NO：75)；
Primer sets D '：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Upper lantern primer LF_D：5’-GGCTAACACCACAATGGCTTTA-3’(SEQ ID NO：76)；
Primer sets E '：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Upper lantern primer LF_E：5’-TTTGCCAATAGCACACCAGTA-3’(SEQ ID NO：77)；
Primer sets F '：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Upper lantern primer LF_F：5’-CCAGGCATAATTTGCCAACGTGC-3’(SEQ ID NO：78)；
Primer sets G '：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_G：5’-ACCCCCAATGGCGTAAACGTT-3’(SEQ ID NO：79)；
Primer sets I '：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_I：5’-TGGGCTGCGTTATTCATAGG-3’(SEQ ID NO：80)；
Primer sets J '：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Upper lantern primer LF_J：5’-GCATTATCAGTCAGTAAGTCACAC-3’(SEQ ID NO：81)；
Primer sets K '：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_K：5’-CGGCACTGGGGTTGTTTATTGAGT-3’(SEQ ID NO：82)；
Primer sets L '：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_L：5’-TTCTGGCTACCAACCGGAAT-3’(SEQ ID NO：83)；
Primer sets M '：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；
Lower lantern primer LB_M：5’-CATATCTTCCGGAGGCGAAA-3’(SEQ ID NO：84)；
Primer sets N '：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；
Lower lantern primer LB_N：5’-CATTGTTTTAGTGATGGGGGC-3’(SEQ ID NO：85)；
Primer sets O '：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Lower lantern primer LB_O：5’-TGGATTATTGATTCCCTGAAACTCC-3’(SEQ ID NO：86)；
Primer sets P '：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_P：5’-ACCCCCAATGGCGTAAACGTT-3’(SEQ ID NO：87)；
Primer sets R '：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_R：5’-TGGGCTGCGTTATTCATAGG-3’(SEQ ID NO：88).
7. the method for claim 1, it is characterised in that in step (2), described enzyme reaction system includes：1×Bst
DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, FIP and BIP of 1.0-1.6mmol/L dNTP, 0.8-2.0 μm of ol/L
Primer, F3 and the B3 primer of 0.15-0.3 μm of ol/L, 0.16-0.64U/ μ L Bst archaeal dna polymerase, the beet of 0-1.5mol/L
Alkali, including or do not include LF or the LB primer of 0.4-1.0 μm of ol/L.
8. the method for claim 1, it is characterised in that the response procedures of described isothermal amplification reactions is：1. 60～65
DEG C hatch 10～90min；2. 80 DEG C terminate reaction 2～20min.
9. for the primer in Constant Temperature Detection yersinia enterocolitica method as claimed in claim 1, it is characterised in that
Described primer includes the primer sets that can expand yersinia enterocolitica genome specificity base sequence, and its sequence is GI
It number is of nucleotide sequence of 169782～171532bp position of the yersinia enterocolitica genome of 123440403
Point or the part of its complementary strand.
10. primer as claimed in claim 9, it is characterised in that described can expand yersinia enterocolitica genome
The primer sets of specific base sequence is selected from any one group of following primer sets A～I；Or be selected from and described primer sets A～I sequence
Or wall scroll sequence homology is 50% and any one group of above primer sets in its complementary strand sequence；
Primer sets A：
Upstream outer primer F3_A：5’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Primer sets B：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Primer sets C：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Primer sets D：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Primer sets E：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Primer sets F：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Primer sets G：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Primer sets H：
Upstream outer primer F3_H：5’-CCATTGGGGGTAACAGTGTT-3’；
Downstream outer primer B3_H：5’-GCAGTGTGCCACCAATCA-3’；
Upstream inner primer FIP_H：5’-CAATCCCTGACAGAGCAGCCAGTTCCGGTCAGACAAACGAC-3’；
Downstream inner primer BIP_H：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’；
Primer sets I：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’.
11. primers as claimed in claim 10, it is characterised in that with in described primer sets A～I sequence or its complementary strand sequence
Wall scroll sequence homology is 50% and above primer sets includes any one group of following primer sets J～R：
Primer sets J：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Primer sets K：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Primer sets L：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Primer sets M：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；
Primer sets N：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；
Primer sets O：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Primer sets P：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Primer sets Q：
Upstream outer primer F3_Q：5’-TAACAGTGTTTCAGCGGCTC-3’；
Downstream outer primer B3_Q：5’-GCAGTGTGCCACCAATCA-3’；
Upstream inner primer FIP_Q：5’-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3’；
Downstream inner primer BIP_Q：5’-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3’；
Primer sets R：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’.
12. primers as claimed in claim 9, it is characterised in that described can expand yersinia enterocolitica genome
The primer sets of specific base sequence also comprises a ring primer；Described ring primer is LF or LB.
13. primers as claimed in claim 12, it is characterised in that described can expand yersinia enterocolitica genome
The primer sets of specific base sequence is selected from following primer sets A ', B ', C ', D ', E ' and, F ', G ', I ', J ' and, K ', L ', M ', N ',
O ', P ', any one group of R '；Or be selected from and described primer sets A ', B ', C ', D ', E ', F ', G ' and, I ', J ', K ', L ' and, M ', N ',
O ', P ', in R ' 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’-TTTGTCTCAGTCAATTTCCC-3’；
Downstream outer primer B3_A：5’-GAAAGCATACATTGGGTGAA-3’；
Upstream inner primer FIP_A：5’-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3’；
Downstream inner primer BIP_A：5’-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3’；
Lower lantern primer LB_A：5’-CAGTTATTGCATTTACTGGTGTGC-3’；
Primer sets B '：
Upstream outer primer F3_B：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_B：5’-GTACTTACCCCTGCATTACGTG-3’；
Upstream inner primer FIP_B：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_B：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_B：5’-CGGCACTGGGGTTGTTTATTGAGT-3’；
Primer sets C '：
Upstream outer primer F3_C：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_C：5’-ACCAATACGGCTAACACC-3’；
Upstream inner primer FIP_C：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_C：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_C：5’-TTCTGGCTACCAACCGGAAT-3’；
Primer sets D '：
Upstream outer primer F3_D：5’-TGGAATTTTGCTTTCTGGC-3’；
Downstream outer primer B3_D：5’-CAAAAGGCATATCCCAGAA-3’；
Upstream inner primer FIP_D：5’-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3’；
Downstream inner primer BIP_D：5’-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3’；
Upper lantern primer LF_D：5’-GGCTAACACCACAATGGCTTTA-3’；
Primer sets E '：
Upstream outer primer F3_E：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_E：5’-AACATCCCAGCAAGAGTG-3’；
Upstream inner primer FIP_E：5’-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_E：5’-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3’；
Upper lantern primer LF_E：5’-TTTGCCAATAGCACACCAGTA-3’；
Primer sets F '：
Upstream outer primer F3_F：5’-TGTCCGAGGCATGAGCTTT-3’；
Downstream outer primer B3_F：5’-GAGCCGCTGAAACACTGTT-3’；
Upstream inner primer FIP_F：5’-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3’；
Downstream inner primer BIP_F：5’-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3’；
Upper lantern primer LF_F：5’-CCAGGCATAATTTGCCAACGTGC-3’；
Primer sets G '：
Upstream outer primer F3_G：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_G：5’-TCGCATCCAACTCAACACC-3’；
Upstream inner primer FIP_G：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_G：5’-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_G：5’-ACCCCCAATGGCGTAAACGTT-3’；
Primer sets I '：
Upstream outer primer F3_I：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_I：5’-CGTTTGGTTGCAAAACAG-3’；
Upstream inner primer FIP_I：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_I：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_I：5’-TGGGCTGCGTTATTCATAGG-3’；
Primer sets J '：
Upstream outer primer F3_J：5’-GGATATGCCTTTTGTCTCAG-3’；
Downstream outer primer B3_J：5’-GAAAGCATACATTGGGTGA-3’；
Upstream inner primer FIP_J：5’-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3’；
Downstream inner primer BIP_J：5’-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3’；
Upper lantern primer LF_J：5’-GCATTATCAGTCAGTAAGTCACAC-3’；
Primer sets K '：
Upstream outer primer F3_K：5’-ATGTTAATGGTTGCAGGGCG-3’；
Downstream outer primer B3_K：5’-CGGGTACTTACCCCTGCATTA-3’；
Upstream inner primer FIP_K：5’-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3’；
Downstream inner primer BIP_K：5’-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3’；
Lower lantern primer LB_K：5’-CGGCACTGGGGTTGTTTATTGAGT-3’；
Primer sets L '：
Upstream outer primer F3_L：5’-GCAGATGCCAATAATGCC-3’；
Downstream outer primer B3_L：5’-CACCACCAATACGGCTAA-3’；
Upstream inner primer FIP_L：5’-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3’；
Downstream inner primer BIP_L：5’-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3’；
Lower lantern primer LB_L：5’-TTCTGGCTACCAACCGGAAT-3’；
Primer sets M '：
Upstream outer primer F3_M：5’-CCTTTTGCTTTCTGTGATTG-3’；
Downstream outer primer B3_M：5’-TATCCCAGAATAAAAACGGC-3’；
Upstream inner primer FIP_M：5’-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3’；
Downstream inner primer BIP_M：5’-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3’；
Lower lantern primer LB_M：5’-CATATCTTCCGGAGGCGAAA-3’；
Primer sets N '：
Upstream outer primer F3_N：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_N：5’-GCAAGAGTGATGATAAATGC-3’；
Upstream inner primer FIP_N：5’-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3’；
Downstream inner primer BIP_N：5’-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3’；
Lower lantern primer LB_N：5’-CATTGTTTTAGTGATGGGGGC-3’；
Primer sets O '：
Upstream outer primer F3_O：5’-CCTTTGTTATTTTATCCGGC-3’；
Downstream outer primer B3_O：5’-AGACACAATAAAGCTCATGC-3’；
Upstream inner primer FIP_O：5’-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3’；
Downstream inner primer BIP_O：5’-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3’；
Lower lantern primer LB_O：5’-TGGATTATTGATTCCCTGAAACTCC-3’；
Primer sets P '：
Upstream outer primer F3_P：5’-GCTATTGGTTGTGGCGTTTG-3’；
Downstream outer primer B3_P：5’-AGCAGCAATCGCATCCAA-3’；
Upstream inner primer FIP_P：5’-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3’；
Downstream inner primer BIP_P：5’-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3’；
Upper lantern primer LF_P：5’-ACCCCCAATGGCGTAAACGTT-3’；
Primer sets R '：
Upstream outer primer F3_R：5’-ATCACTTTTGATGGTACGC-3’；
Downstream outer primer B3_R：5’-ACTGTATGCCGTTTGGTT-3’；
Upstream inner primer FIP_R：5’-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3’；
Downstream inner primer BIP_R：5’-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3’；
Lower lantern primer LB_R：5’-TGGGCTGCGTTATTCATAGG-3’.
14. 1 kinds of kits for Constant Temperature Detection yersinia enterocolitica, it is characterised in that described kit includes
Primer as described in any one of claim 9～13.
15. kits as claimed in claim 14, it is characterised in that its also include Bst DNA polymerase reaction buffer solution,
Bst archaeal dna polymerase, dNTP solution, Mg2+, one or more in glycine betaine.
16. 1 kinds of kits for Constant Temperature Detection yersinia enterocolitica, it is characterised in that the enzyme of described kit
Reaction system includes：1 × Bst DNA polymerase reaction buffer solution, 2-9mmol/L Mg2+, 1.0-1.6mmol/L dNTP, 0.8-
FIP and the BIP primer of 2.0 μm of ol/L, F3 and the B3 primer of 0.15-0.3 μm of ol/L, including or do not include 0.4-1.0 μm of ol/L
LF or LB primer, 0.16-0.64U/ μ L Bst archaeal dna polymerase, and the glycine betaine of 0-1.5mol/L.
17. 1 kinds of carriers, it is characterised in that described carrier comprises the primer as described in any one of claim 9～13.
Application in Constant Temperature Detection yersinia enterocolitica for 18. primers, it is characterised in that described primer is for such as right
Require the primer described in any one of 9～13.
19. kits as described in any one of claim 14～16 or carrier as claimed in claim 17 are at constant temperature small intestine
Application in colitis Yersinia ruckeri.
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| CN202010017857.XA CN111073987B (en) | 2016-08-30 | 2016-08-30 | Rapid constant-temperature detection method, primer group and kit for yersinia enterocolitica |
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| CN107663545A (en) * | 2017-09-19 | 2018-02-06 | 温和心 | Detect primer sets and the application of yersinia enterocolitica |
| CN110734992A (en) * | 2019-11-27 | 2020-01-31 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | LAMP (loop-mediated isothermal amplification) detection kit for food-borne enterocolitis yersinia and application of LAMP detection kit |
| CN112795669A (en) * | 2020-12-30 | 2021-05-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107663545A (en) * | 2017-09-19 | 2018-02-06 | 温和心 | Detect primer sets and the application of yersinia enterocolitica |
| CN110734992A (en) * | 2019-11-27 | 2020-01-31 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | LAMP (loop-mediated isothermal amplification) detection kit for food-borne enterocolitis yersinia and application of LAMP detection kit |
| CN112795669A (en) * | 2020-12-30 | 2021-05-14 | 广东省微生物研究所（广东省微生物分析检测中心） | Yersinia enterocolitica standard strain containing specific molecular target and detection and application thereof |
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| CN111073987B (en) | 2022-09-20 |
| CN106434890B (en) | 2020-02-21 |
| CN111073987A (en) | 2020-04-28 |
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