CN111073987B - Rapid constant-temperature detection method, primer group and kit for yersinia enterocolitica - Google Patents
Rapid constant-temperature detection method, primer group and kit for yersinia enterocolitica Download PDFInfo
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Abstract
The invention discloses a rapid constant-temperature detection method, a primer group and a kit for yersinia enterocolitica. The method comprises the following steps: extracting genome DNA from a sample to be detected; taking the genome DNA as a template, taking a primer group capable of amplifying a specific sequence of the yersinia enterocolitica as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system; and determining whether the sample to be detected has the yersinia enterocolitica or not by judging whether the reaction result is positive or not. The detection method has the advantages of high sensitivity and high specificity, short detection time, simple result judgment, convenient operation, low cost and wide application prospect.
Description
The application is filed on 2016, 8, 30, and has the application number of 201610767671.X and the name of the invention as follows: the patent application of the Chinese invention of 'a method, a primer and a kit for rapidly detecting yersinia enterocolitica at constant temperature' is divided into separate cases.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting yersinia enterocolitica at a constant temperature.
Background
Yersinia enterocolitica (Yersinia enterocolitica) is widely distributed in the natural world, is one of a few enteropathogenic bacteria capable of growing at refrigeration temperature, and can cause diseases such as arthritis, erythema nodosum and mesenteric lymphadenitis besides gastrointestinal symptoms, even septicemia and death. The clinical symptoms of the bacteria after infection are often not obvious, and misdiagnosis is easily caused. Because the bacteria can survive in a low-temperature environment, food stored in a refrigerator is an important infection source for the bacteria infection in modern society, and the bacteria are listed as routine detection items of import and export food in many countries.
At present, bacteria in food are generally detected by using a traditional physiological and biochemical method, and due to the long detection period, the operation is relatively complex and the detection efficiency is relatively low, the requirements of high flux, high sensitivity, high specificity, rapidness and convenience in the detection process of food-borne pathogenic bacteria in modern society are difficult to meet. In recent years, with the development of nucleic acid molecule detection technology, researchers have developed detection means such as PCR and real-time fluorescence PCR, but both methods require special detection instruments, and are not suitable for real-time field detection widely used in basic detection departments, especially in production lines of enterprises. In order to ensure the safety of food, a rapid, simple and accurate method for detecting yersinia enterocolitica in food is urgently needed.
Loop-mediated isothermal amplification (LAMP) is a novel isothermal Nucleic acid amplification method developed in recent years, which designs 4 specific primers (including upstream and downstream outer primers F3 and B3, FIP consisting of F1C and F2, and upstream and downstream inner primers FIP and BIP consisting of B1C and B2) for 6 regions of a target sequence, and completes the Nucleic acid amplification reaction by incubating for about 60min at an isothermal condition using a DNA polymerase having a strand displacement activity, and generates a macroscopic reaction byproduct, namely, white magnesium pyrophosphate precipitate (see Notomi T, Oyayaama H, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification (DNA of Research, Jeans, 2000; 3612, 63, Jeans, 3628). The technology can be completed at a constant temperature without a PCR instrument or a fluorescent quantitative PCR instrument, can judge the reaction result by naked eyes, and has the advantages of high sensitivity, strong specificity, short reaction time, convenient operation, low cost and the like.
Primer design is the most critical step in LAMP technology, and the conventional method is to introduce the acknowledged specific gene of a certain organism to be detected into an online website (http:// primer explorer. jp/e) designed by LAMP primers, and set relevant parameters to generate a primer group. That is, the user must first ensure that the target gene is a specific sequence for the species being tested. Taking the patent CN 101182574B as an example, the LAMP technology is adopted to detect the yersinia enterocolitica aiming at the sequence of the 16S-23S region which is a specific region of the yersinia enterocolitica reported in the literature. However, the so-called "recognized specific genes" are often based on a delayed knowledge and are not necessarily updated based on the ever-increasing genome data of microorganisms, so that primers obtained based on the target gene sequences are not necessarily able to ensure their versatility and/or specificity in practical applications. Table 1 of the present invention shows the problem that the versatility is not ensured in the prior art. That is, the yersinia enterocolitica detection sequence used in the prior art method is not actually common to yersinia enterocolitica, i.e., there is a possibility that a partial strain of yersinia enterocolitica may be overlooked. A similar problem exists in the confirmation of specificity, i.e., there is a possibility that yersinia non-enterocolitis may be mistakenly identified as yersinia enterocolitis. Therefore, a yersinia enterocolitica detection method capable of ensuring specificity and universality is urgently needed in the industry, and simultaneously, the requirements of basic detection departments on rapidness and convenience are met, and real-time field detection can be conveniently developed in an enterprise production line.
Disclosure of Invention
The invention aims to overcome the defects of insufficient primer universality and specificity in the primer design of the prior LAMP technology, fully utilizes abundant microbial genome sequence information in the current public data resources and corresponding sequence analysis tools, designs a primer group for specifically identifying the yersinia enterocolitica, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a Yersinia enterocolitica LAMP primer based on microbial genome data resources (data obtained by 8/5/2013) in a GenBank database, and provides a method, a primer group and a kit for rapid isothermal amplification detection of Yersinia enterocolitica. The detection method for detecting the yersinia enterocolitica has the advantages of high sensitivity and specificity, short detection time, simple result judgment, convenience in operation and low cost.
The invention provides a method for rapidly detecting yersinia enterocolitica strains, which comprises the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) carrying out constant-temperature amplification reaction under an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the yersinia enterocolitica genome as a primer;
(3) and determining whether the sample to be detected has the yersinia enterocolitica or not by judging whether the reaction result is positive or not.
The method for detecting the yersinia enterocolitica strain at constant temperature comprises the steps of extracting genome DNA from a sample to be detected, carrying out constant-temperature amplification reaction by using the genome DNA as a template and a specific amplification primer group of the yersinia enterocolitica as primers, and then determining whether the sample to be detected contains the yersinia enterocolitica or not by judging whether the reaction result is positive or not. Wherein, the enzyme reaction system includes but is not limited to DNA polymerase reaction system.
In the invention, the genome-specific base sequence of the Yersinia enterocolitica is the bit sequence of 169782-171532 bp of the Yersinia enterocolitica with the GI number of 123440403.
In the present invention, the primer set capable of amplifying a nucleotide sequence specific to a Yersinia enterocolitica genome is a part of a nucleic acid sequence of 169782 to 171532bp positions of the genome (GI No. 123440403) or a part of a complementary strand thereof. Wherein the Yersinia enterocolitis genome-specific base sequence is a base sequence which is unique to the Yersinia enterocolitis genome and is not contained in the genome of another microorganism.
Wherein the primer group capable of amplifying the specific base sequence of the Yersinia enterocolitica genome includes, but is not limited to, any one selected from the following primer groups A to I, or any one selected from the primer groups having a homology of 50% or more with a single sequence in the sequence of the primer group or the complementary strand sequence thereof.
Primer set 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);
the downstream inner primer BIP _ A: 5'-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3' (SEQ ID NO: 4);
primer set 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);
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3' (SEQ ID NO: 8);
primer set 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);
the downstream inner primer BIP _ C: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3' (SEQ ID NO: 12);
primer set 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);
the downstream inner primer BIP _ D: 5'-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3' (SEQ ID NO: 16);
primer set 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);
the downstream inner primer BIP _ E: 5'-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3' (SEQ ID NO: 20);
a primer set 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);
the downstream inner primer BIP _ F: 5'-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3' (SEQ ID NO: 24);
primer set 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);
the downstream inner primer BIP _ G: 5 '-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3' (SEQ ID NO: 28);
a primer set 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);
the downstream inner primer BIP _ H: 5'-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3' (SEQ ID NO: 32);
primer set 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);
the downstream inner primer BIP _ I: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3' (SEQ ID NO: 36).
In the present invention, the primer set capable of amplifying the specific base sequence of the yersinia enterocolitica genome may further include a primer set having a homology of 50% or more with a single sequence in the sequences of each of the aforementioned primer sets or the complementary strand sequences thereof, and the primer set includes, but is not limited to, any one of the following primer sets J to R:
primer set J:
upstream outer primer F3_ J: 5'-GGATATGCCTTTTGTCTCAG-3' (SEQ ID NO: 37) (50% homology to primer F3_ A5'-TTTGTCTCAGTCAATTTCCC-3');
downstream outer primer B3_ J: 5'-GAAAGCATACATTGGGTGA-3' (SEQ ID NO: 38);
upstream inner primer FIP _ J: 5'-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3' (SEQ ID NO: 39);
the downstream inner primer BIP _ J: 5'-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3' (SEQ ID NO: 40);
primer set 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) (81.8% homology to primer B3_ B5 '-GTACTTACCCCTGCATTACGTG-3');
upstream inner primer FIP _ K: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3' (SEQ ID NO: 43);
the downstream inner primer BIP _ K: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3' (SEQ ID NO: 44);
a primer set 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) (77.8% homology to primer B3_ C5'-ACCAATACGGCTAACACC-3');
upstream inner primer FIP _ L: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3' (SEQ ID NO: 47);
the downstream inner primer BIP _ L: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3' (SEQ ID NO: 48);
and (3) primer group 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) (52.6% homology to primer B3_ D5'-CAAAAGGCATATCCCAGAA-3');
upstream inner primer FIP _ M: 5'-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3' (SEQ ID NO: 51);
the downstream inner primer BIP _ M: 5'-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3' (SEQ ID NO: 52);
a primer set 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) (50% homology to primer B3_ E5'-AACATCCCAGCAAGAGTG-3');
upstream inner primer FIP _ N: 5'-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3' (SEQ ID NO: 55);
the downstream inner primer BIP _ N: 5'-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3' (SEQ ID NO: 56);
a primer group 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) (57.9% homology to the complementary strand 5'-AAAGCTCATGCCTCGGACA-3' of primer F3_ F);
upstream inner primer FIP _ O: 5'-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3' (SEQ ID NO: 59);
the downstream inner primer BIP _ O: 5'-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3' (SEQ ID NO: 60);
a primer group 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) (52.6% homology to primer B3_ G5'-TCGCATCCAACTCAACACC-3');
upstream inner primer FIP _ P: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3' (SEQ ID NO: 63);
a downstream inner primer BIP _ P: 5'-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3' (SEQ ID NO: 64);
a primer set Q:
upstream outer primer F3_ Q: 5'-TAACAGTGTTTCAGCGGCTC-3' (SEQ ID NO: 65) (50% homology to primer F3_ H5'-CCATTGGGGGTAACAGTGTT-3');
downstream outer primer B3_ Q: 5'-GCAGTGTGCCACCAATCA-3' (SEQ ID NO: 66);
upstream inner primer FIP _ Q: 5'-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3' (SEQ ID NO: 67);
the downstream inner primer BIP _ Q: 5'-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3' (SEQ ID NO: 68);
a primer set 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) (50% homology to primer B3_ I5'-CGTTTGGTTGCAAAACAG-3');
upstream inner primer FIP _ R: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3' (SEQ ID NO: 71);
the downstream inner primer BIP _ R: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3' (SEQ ID NO: 72).
In the method of the present invention, the primer set capable of amplifying the genome-specific base sequence of yersinia enterocolitica may include, but is not limited to, a single loop primer. Preferably, the loop primer may be one, including loop primer LF or LB. The primer group capable of amplifying the genome-specific base sequence of the yersinia enterocolitica is selected from any one of the following primer groups A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R'; or any one selected from the group consisting of primer sets having a homology of 50% or more with respect to a single sequence in the sequences of the primer sets A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-TTTGTCTCAGTCAATTTCCC-3', respectively;
downstream outer primer B3_ a: 5'-GAAAGCATACATTGGGTGAA-3', respectively;
upstream inner primer FIP _ A: 5'-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3', respectively;
the downstream inner primer BIP _ A: 5'-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3';
downstream loop primer LB _ a: 5'-CAGTTATTGCATTTACTGGTGTGC-3' (SEQ ID NO: 73);
a primer set B':
upstream outer primer F3_ B: 5'-ATGTTAATGGTTGCAGGGCG-3', respectively;
downstream outer primer B3_ B: 5'-GTACTTACCCCTGCATTACGTG-3', respectively;
upstream inner primer FIP _ B: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3', respectively;
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
downstream loop primer LB _ B: 5'-CGGCACTGGGGTTGTTTATTGAGT-3' (SEQ ID NO: 74);
a primer set C':
upstream outer primer F3_ C: 5'-GCAGATGCCAATAATGCC-3', respectively;
downstream outer primer B3 — C: 5'-ACCAATACGGCTAACACC-3', respectively;
upstream inner primer FIP _ C: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ C: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3', respectively;
downstream loop primer LB _ C: 5'-TTCTGGCTACCAACCGGAAT-3' (SEQ ID NO: 75);
a primer set D':
the upstream outer primer F3_ D: 5'-TGGAATTTTGCTTTCTGGC-3', respectively;
downstream outer primer B3_ D: 5'-CAAAAGGCATATCCCAGAA-3', respectively;
upstream inner primer FIP _ D: 5'-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3', respectively;
the downstream inner primer BIP _ D: 5'-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3', respectively;
upstream loop primer LF _ D: 5'-GGCTAACACCACAATGGCTTTA-3' (SEQ ID NO: 76);
a primer set E':
upstream outer primer F3_ E: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3_ E: 5'-AACATCCCAGCAAGAGTG-3', respectively;
upstream inner primer FIP _ E: 5'-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3';
the downstream inner primer BIP _ E: 5'-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3', respectively;
upstream loop primer LF _ E: 5'-TTTGCCAATAGCACACCAGTA-3' (SEQ ID NO: 77);
a primer set F':
upstream outer primer F3 — F: 5'-TGTCCGAGGCATGAGCTTT-3', respectively;
downstream outer primer B3 — F: 5'-GAGCCGCTGAAACACTGTT-3', respectively;
upstream inner primer FIP _ F: 5'-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3', respectively;
the downstream inner primer BIP _ F: 5'-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3', respectively;
upstream loop primer LF _ F: 5'-CCAGGCATAATTTGCCAACGTGC-3' (SEQ ID NO: 78);
a primer set G':
upstream outer primer F3_ G: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ G: 5'-TCGCATCCAACTCAACACC-3', respectively;
upstream inner primer FIP _ G: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ G: 5 '-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3';
upstream loop primer LF _ G: 5'-ACCCCCAATGGCGTAAACGTT-3' (SEQ ID NO: 79);
a primer set I':
upstream outer primer F3_ I: 5'-ATCACTTTTGATGGTACGC-3', respectively;
downstream outer primer B3 — I: 5'-CGTTTGGTTGCAAAACAG-3', respectively;
an upstream inner primer FIP _ I: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3', respectively;
the downstream inner primer BIP _ I: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3', respectively;
downstream loop primer LB _ I: 5'-TGGGCTGCGTTATTCATAGG-3' (SEQ ID NO: 80);
primer set J':
upstream outer primer F3_ J: 5'-GGATATGCCTTTTGTCTCAG-3', respectively;
downstream outer primer B3_ J: 5'-GAAAGCATACATTGGGTGA-3', respectively;
upstream inner primer FIP _ J: 5'-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3', respectively;
the downstream inner primer BIP _ J: 5'-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3', respectively;
upstream loop primer LF _ J: 5'-GCATTATCAGTCAGTAAGTCACAC-3' (SEQ ID NO: 81);
a primer set K':
upstream outer primer F3_ K: 5'-ATGTTAATGGTTGCAGGGCG-3', respectively;
downstream outer primer B3_ K: 5'-CGGGTACTTACCCCTGCATTA-3';
an upstream inner primer FIP _ K: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3', respectively;
the downstream inner primer BIP _ K: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
downstream loop primer LB _ K: 5'-CGGCACTGGGGTTGTTTATTGAGT-3' (SEQ ID NO: 82);
a primer set L':
upstream outer primer F3_ L: 5'-GCAGATGCCAATAATGCC-3', respectively;
downstream outer primer B3_ L: 5'-CACCACCAATACGGCTAA-3', respectively;
upstream inner primer FIP _ L: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ L: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3', respectively;
downstream loop primer LB _ L: 5'-TTCTGGCTACCAACCGGAAT-3' (SEQ ID NO: 83);
a primer set M':
upstream outer primer F3_ M: 5'-CCTTTTGCTTTCTGTGATTG-3', respectively;
downstream outer primer B3_ M: 5'-TATCCCAGAATAAAAACGGC-3', respectively;
upstream inner primer FIP _ M: 5'-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3', respectively;
the downstream inner primer BIP _ M: 5'-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3', respectively;
downstream loop primer LB _ M: 5'-CATATCTTCCGGAGGCGAAA-3' (SEQ ID NO: 84);
a primer set N':
upstream outer primer F3 — N: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3 — N: 5'-GCAAGAGTGATGATAAATGC-3', respectively;
upstream inner primer FIP _ N: 5'-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3', respectively;
the downstream inner primer BIP _ N: 5'-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3', respectively;
downstream loop primer LB _ N: 5'-CATTGTTTTAGTGATGGGGGC-3' (SEQ ID NO: 85);
a primer set O':
upstream outer primer F3 — O: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3 — O: 5'-AGACACAATAAAGCTCATGC-3', respectively;
upstream inner primer FIP _ O: 5'-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3', respectively;
the downstream inner primer BIP _ O: 5'-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3', respectively;
downstream loop primer LB _ O: 5'-TGGATTATTGATTCCCTGAAACTCC-3' (SEQ ID NO: 86);
a primer set P':
upstream outer primer F3_ P: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ P: 5'-AGCAGCAATCGCATCCAA-3', respectively;
upstream inner primer FIP _ P: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ P: 5'-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3', respectively;
upstream loop primer LF _ P: 5'-ACCCCCAATGGCGTAAACGTT-3' (SEQ ID NO: 87);
a primer set R':
upstream outer primer F3_ R: 5'-ATCACTTTTGATGGTACGC-3', respectively;
downstream outer primer B3_ R: 5'-ACTGTATGCCGTTTGGTT-3', respectively;
upstream inner primer FIP _ R: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3', respectively;
the downstream inner primer BIP _ R: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3', respectively;
downstream loop primer LB _ R: 5'-TGGGCTGCGTTATTCATAGG-3' (SEQ ID NO: 88).
In a specific embodiment (including a loop primer), the enzyme reaction system for isothermal amplification is as follows: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.4-1.0 mu mol/L LF or LB primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. In another embodiment (without loop primer), the enzyme reaction system for isothermal amplification is: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. The loop primer contributes to improvement of reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at 60-65 ℃ for 10-90 min, preferably 10-60 min; ② terminating the reaction for 2-20 min at 80 ℃. The invention is not limited to the implementation of the detection method of the invention by other suitable reaction procedures.
In the method of the present invention, the detection method includes, but is not limited to, electrophoresis detection, turbidity detection, color detection, or the like. The electrophoresis detection is preferably a gel electrophoresis detection method, and may be agarose gel or polyacrylamide gel. In the electrophoresis detection result, if the electrophoresis image shows a characteristic stepped strip, the sample to be detected is positive to the yersinia enterocolitica and contains the yersinia enterocolitica; if the electrophoretogram does not present the characteristic ladder-shaped strip, the sample to be tested is negative to Yersinia enterocolitica. The turbidity detection is to detect turbidity by visual observation or a turbidity meter, and if the detection tube is obviously turbid, the sample to be detected is positive to yersinia enterocolitica and contains yersinia enterocolitica; if no turbidity is found, the sample to be tested is negative to the yersinia enterocolitica. Or the bottom of the reaction tube can be visually observed whether the reaction tube is precipitated or not after centrifugation, if the reaction tube is precipitated, the sample to be detected is positive to the enterocolitis yersinia and contains the enterocolitis yersinia; if no precipitate is formed at the bottom of the reaction tube, the sample to be detected is negative to the yersinia enterocolitica.
The color development detection is to add color development reagent including but not limited to calcein (50 μ M) or SYBR Green I (30-50X), or hydroxyl naphthol blue (i.e. HNB, 120-ion 150 μ M) into the reaction tube. When calcein or SYBR Green I is adopted as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to the yersinia enterocolitica; if the color after the reaction is green, the sample to be detected is positive to the yersinia enterocolitica and contains the yersinia enterocolitica. When hydroxyl naphthol blue is used as a color developing agent, if the color after reaction is violet, the sample to be detected is negative to enterocolitis yersinia; and if the color after the reaction is sky blue, the sample to be detected is positive for the yersinia enterocolitica. The chromogenic detection can be used for detecting the reaction result in real time or at the end point through a detection instrument besides observing the reaction result through naked eyes, and by reasonably setting the threshold value of the negative reaction, when the reaction result of the sample to be detected is lower than or equal to the threshold value, the sample to be detected is negative to the yersinia enterocolitica; and when the reaction result of the sample to be detected is greater than the threshold value, determining that the sample to be detected is positive for the yersinia enterocolitica. The detection instrument comprises but is not limited to a fluorescence spectrophotometer, a fluorescence quantitative PCR instrument, a constant temperature amplification microfluidic chip nucleic acid analyzer, a Genie II isothermal amplification fluorescence detection system and the like.
In the color development detection, if calcein or hydroxynaphthol blue is used as a color developing agent, the color developing agent can be added before the constant-temperature amplification reaction, or can be added after the constant-temperature amplification reaction is completed, preferably before the constant-temperature amplification reaction, so that the possibility of reaction pollution can be effectively reduced. If SYBR Green I is adopted as a color developing agent, the SYBR Green I is added after the isothermal amplification reaction is finished. If calcein is used as color-developing agent, adding 50 μ M calcein into enzyme reaction system, and adding 0.6-1mM [ Mn ] 2+ ]For example, 0.6-1mM MnCl 2 。
The invention also provides a primer used in the method for detecting the yersinia enterocolitica strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of Yersinia enterocolitica genome, and the primer comprises but is not limited to a part of nucleic acid sequence of 169782-171532 bp position of Yersinia enterocolitica genome with GI number of 123440403 or a part of complementary strand thereof.
Wherein the primer group capable of amplifying the Yersinia enterocolitica genome-specific base sequence is selected from any one of the following primer groups, or from any one of the primer groups having a homology of 50% or more with a single sequence in the sequences of the primer groups or the complementary strand sequences thereof. Wherein the primer set includes, but is not limited to, any one of the following primer sets A to I. The primer set having a homology of 50% or more with respect to a single sequence in the aforementioned primer set sequence or the complementary strand sequence thereof includes, but is not limited to, any one of the following primer sets J to R.
Primer set a:
upstream outer primer F3_ a: 5'-TTTGTCTCAGTCAATTTCCC-3', respectively;
downstream outer primer B3_ a: 5'-GAAAGCATACATTGGGTGAA-3', respectively;
upstream inner primer FIP _ A: 5'-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3', respectively;
the downstream inner primer BIP _ A: 5'-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-ATGTTAATGGTTGCAGGGCG-3', respectively;
downstream outer primer B3_ B: 5'-GTACTTACCCCTGCATTACGTG-3', respectively;
upstream inner primer FIP _ B: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3';
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
primer set C:
upstream outer primer F3_ C: 5'-GCAGATGCCAATAATGCC-3', respectively;
downstream outer primer B3 — C: 5'-ACCAATACGGCTAACACC-3', respectively;
upstream inner primer FIP _ C: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ C: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3', respectively;
primer set D:
upstream outer primer F3_ D: 5'-TGGAATTTTGCTTTCTGGC-3', respectively;
downstream outer primer B3_ D: 5'-CAAAAGGCATATCCCAGAA-3', respectively;
upstream inner primer FIP _ D: 5'-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3', respectively;
the downstream inner primer BIP _ D: 5'-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3', respectively;
primer set E:
upstream outer primer F3_ E: 5'-CCTTTGTTATTTTATCCGGC-3';
downstream outer primer B3_ E: 5'-AACATCCCAGCAAGAGTG-3', respectively;
upstream inner primer FIP _ E: 5'-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3';
the downstream inner primer BIP _ E: 5'-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3';
a primer set F:
upstream outer primer F3 — F: 5'-TGTCCGAGGCATGAGCTTT-3', respectively;
downstream outer primer B3 — F: 5'-GAGCCGCTGAAACACTGTT-3', respectively;
upstream inner primer FIP _ F: 5'-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3';
the downstream inner primer BIP _ F: 5'-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3', respectively;
primer set G:
upstream outer primer F3_ G: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ G: 5'-TCGCATCCAACTCAACACC-3', respectively;
upstream inner primer FIP _ G: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ G: 5 '-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3';
a primer set H:
upstream outer primer F3 — H: 5'-CCATTGGGGGTAACAGTGTT-3', respectively;
downstream outer primer B3 — H: 5'-GCAGTGTGCCACCAATCA-3', respectively;
upstream inner primer FIP _ H: 5'-CAATCCCTGACAGAGCAGCCAGTTCCGGTCAGACAAACGAC-3';
the downstream inner primer BIP _ H: 5'-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3', respectively;
primer set I:
upstream outer primer F3_ I: 5'-ATCACTTTTGATGGTACGC-3', respectively;
downstream outer primer B3 — I: 5'-CGTTTGGTTGCAAAACAG-3', respectively;
upstream inner primer FIP _ I: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3', respectively;
the downstream inner primer BIP _ I: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3', respectively;
primer set J:
upstream outer primer F3_ J: 5'-GGATATGCCTTTTGTCTCAG-3', respectively;
downstream outer primer B3_ J: 5'-GAAAGCATACATTGGGTGA-3', respectively;
upstream inner primer FIP _ J: 5'-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3', respectively;
a downstream inner primer BIP _ J: 5'-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3', respectively;
primer set K:
upstream outer primer F3_ K: 5'-ATGTTAATGGTTGCAGGGCG-3';
downstream outer primer B3_ K: 5'-CGGGTACTTACCCCTGCATTA-3', respectively;
an upstream inner primer FIP _ K: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3', respectively;
the downstream inner primer BIP _ K: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
a primer set L:
upstream outer primer F3_ L: 5'-GCAGATGCCAATAATGCC-3', respectively;
downstream outer primer B3_ L: 5'-CACCACCAATACGGCTAA-3', respectively;
upstream inner primer FIP _ L: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ L: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3', respectively;
and (3) primer group M:
upstream outer primer F3_ M: 5'-CCTTTTGCTTTCTGTGATTG-3', respectively;
downstream outer primer B3_ M: 5'-TATCCCAGAATAAAAACGGC-3', respectively;
upstream inner primer FIP _ M: 5'-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3', respectively;
the downstream inner primer BIP _ M: 5'-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3', respectively;
primer set N:
upstream outer primer F3 — N: 5'-CCTTTGTTATTTTATCCGGC-3';
downstream outer primer B3 — N: 5'-GCAAGAGTGATGATAAATGC-3', respectively;
upstream inner primer FIP _ N: 5'-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3', respectively;
the downstream inner primer BIP _ N: 5'-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3', respectively;
a primer group O:
upstream outer primer F3 — O: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3 — O: 5'-AGACACAATAAAGCTCATGC-3', respectively;
upstream inner primer FIP _ O: 5'-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3', respectively;
the downstream inner primer BIP _ O: 5'-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3', respectively;
a primer group P:
upstream outer primer F3_ P: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ P: 5'-AGCAGCAATCGCATCCAA-3', respectively;
upstream inner primer FIP _ P: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ P: 5'-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3';
a primer set Q:
upstream outer primer F3_ Q: 5'-TAACAGTGTTTCAGCGGCTC-3', respectively;
downstream outer primer B3_ Q: 5'-GCAGTGTGCCACCAATCA-3', respectively;
upstream inner primer FIP _ Q: 5'-AGAGCAGCCAGAGTGCTCGATGGGTTCCGGTCAGACAAAC-3', respectively;
the downstream inner primer BIP _ Q: 5'-TACACCTCCGCAGGTTATGCATTGCGACAGCAGCAATCGCAT-3', respectively;
a primer set R:
upstream outer primer F3_ R: 5'-ATCACTTTTGATGGTACGC-3', respectively;
downstream outer primer B3 — R: 5'-ACTGTATGCCGTTTGGTT-3', respectively;
an upstream inner primer FIP _ R: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3';
the downstream inner primer BIP _ R: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3' are provided.
In the primer used in the method for detecting yersinia enterocolitica at constant temperature, the primer group capable of amplifying the specific base sequence of the yersinia enterocolitica genome can further comprise, but is not limited to, a loop primer; preferably, the loop primer is one, including LF or LB. The primer group capable of amplifying the genome-specific base sequence of the yersinia enterocolitica is selected from any one of the following primer groups A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R'; or any one selected from the group consisting of primer sets having a homology of 50% or more with respect to a single sequence in the sequences of the primer sets A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-TTTGTCTCAGTCAATTTCCC-3', respectively;
downstream outer primer B3_ a: 5'-GAAAGCATACATTGGGTGAA-3', respectively;
upstream inner primer FIP _ A: 5'-TAACAAAGGTCATGCCCACAGTTTGGTGTGACTTACTGACT-3', respectively;
the downstream inner primer BIP _ A: 5'-CGGCATTGATTTATCTGTCGGTTAAGTGCCGATTAGTTTTGC-3', respectively;
downstream loop primer LB _ a: 5'-CAGTTATTGCATTTACTGGTGTGC-3', respectively;
a primer set B':
upstream outer primer F3_ B: 5'-ATGTTAATGGTTGCAGGGCG-3', respectively;
downstream outer primer B3_ B: 5'-GTACTTACCCCTGCATTACGTG-3', respectively;
upstream inner primer FIP _ B: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3', respectively;
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
downstream loop primer LB _ B: 5'-CGGCACTGGGGTTGTTTATTGAGT-3', respectively;
a primer set C':
upstream outer primer F3_ C: 5'-GCAGATGCCAATAATGCC-3';
downstream outer primer B3 — C: 5'-ACCAATACGGCTAACACC-3', respectively;
upstream inner primer FIP _ C: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ C: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3';
downstream loop primer LB _ C: 5'-TTCTGGCTACCAACCGGAAT-3', respectively;
a primer set D':
upstream outer primer F3_ D: 5'-TGGAATTTTGCTTTCTGGC-3', respectively;
downstream outer primer B3_ D: 5'-CAAAAGGCATATCCCAGAA-3', respectively;
upstream inner primer FIP _ D: 5'-TGAGATCATCGGTGACTGCACAACCGGAATTCAATCTGG-3', respectively;
the downstream inner primer BIP _ D: 5'-CATATCTTCCGGAGGCGAAACGATAGTAATCAGCAAAGGA-3', respectively;
upstream loop primer LF _ D: 5'-GGCTAACACCACAATGGCTTTA-3', respectively;
a primer set E':
upstream outer primer F3_ E: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3_ E: 5'-AACATCCCAGCAAGAGTG-3', respectively;
upstream inner primer FIP _ E: 5'-TGGGTGAATGCCATAAGTGCCTGTCGGTTCAGTTATTGC-3';
a downstream inner primer BIP _ E: 5'-CGATGTTTGGAGCGTTCATGTGATAAATGCGGGGAGTTT-3', respectively;
upstream loop primer LF _ E: 5'-TTTGCCAATAGCACACCAGTA-3', respectively;
a primer set F':
upstream outer primer F3 — F: 5'-TGTCCGAGGCATGAGCTTT-3', respectively;
downstream outer primer B3 — F: 5'-GAGCCGCTGAAACACTGTT-3', respectively;
upstream inner primer FIP _ F: 5'-GCAAGCAGTGTAAATCGTCCACCTCCCATTGATCACCCGATCT-3', respectively;
the downstream inner primer BIP _ F: 5'-GTGGCGTTTGGTATTTTGCTGGCCCCCAATGGCGTAAACGTTA-3';
upstream loop primer LF _ F: 5'-CCAGGCATAATTTGCCAACGTGC-3', respectively;
a primer set G':
upstream outer primer F3_ G: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ G: 5'-TCGCATCCAACTCAACACC-3', respectively;
upstream inner primer FIP _ G: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ G: 5 '-CTATCGAGCACTCTGGCTGCTC-CTGGCCGCCAATGCATAA-3';
upstream loop primer LF _ G: 5'-ACCCCCAATGGCGTAAACGTT-3', respectively;
a primer set I':
upstream outer primer F3_ I: 5'-ATCACTTTTGATGGTACGC-3', respectively;
downstream outer primer B3 — I: 5'-CGTTTGGTTGCAAAACAG-3', respectively;
upstream inner primer FIP _ I: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3', respectively;
the downstream inner primer BIP _ I: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3', respectively;
downstream loop primer LB _ I: 5'-TGGGCTGCGTTATTCATAGG-3', respectively;
primer set J':
upstream outer primer F3_ J: 5'-GGATATGCCTTTTGTCTCAG-3', respectively;
downstream outer primer B3_ J: 5'-GAAAGCATACATTGGGTGA-3', respectively;
upstream inner primer FIP _ J: 5'-AACAAAGGTCATGCCCACAGTTTCTTCCACCCGAGTTT-3', respectively;
the downstream inner primer BIP _ J: 5'-CGGCGGCATTGATTTATCTGTGCCGATTAGTTTTGCCAAT-3', respectively;
upstream loop primer LF _ J: 5'-GCATTATCAGTCAGTAAGTCACAC-3', respectively;
a primer set K':
upstream outer primer F3_ K: 5'-ATGTTAATGGTTGCAGGGCG-3';
downstream outer primer B3_ K: 5'-CGGGTACTTACCCCTGCATTA-3', respectively;
an upstream inner primer FIP _ K: 5'-CAAGGTGCTGCTGCCTAACTCTCCGAAGGCCAGATTGTCAC-3', respectively;
the downstream inner primer BIP _ K: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3', respectively;
downstream loop primer LB _ K: 5'-CGGCACTGGGGTTGTTTATTGAGT-3', respectively;
a primer set L':
upstream outer primer F3_ L: 5'-GCAGATGCCAATAATGCC-3', respectively;
downstream outer primer B3_ L: 5'-CACCACCAATACGGCTAA-3', respectively;
upstream inner primer FIP _ L: 5'-CAAAAGGTTAAAACGCCCGCAGTTGGATGCTATTTTGGC-3', respectively;
the downstream inner primer BIP _ L: 5'-CAGGGTATGAATACTGGAATTTTGCCAATGGCTTTAAGAACCAGA-3', respectively;
downstream loop primer LB _ L: 5'-TTCTGGCTACCAACCGGAAT-3', respectively;
a primer set M':
upstream outer primer F3_ M: 5'-CCTTTTGCTTTCTGTGATTG-3', respectively;
downstream outer primer B3_ M: 5'-TATCCCAGAATAAAAACGGC-3', respectively;
upstream inner primer FIP _ M: 5'-GCTTTAAGAACCAGATTGAATTCCGTCAGGGTATGAATACTGGAA-3', respectively;
the downstream inner primer BIP _ M: 5'-TGCAGTCACCGATGATCTCACAGCAAAGGAATATTACGCT-3', respectively;
downstream loop primer LB _ M: 5'-CATATCTTCCGGAGGCGAAA-3', respectively;
a primer set N':
upstream outer primer F3 — N: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3 — N: 5'-GCAAGAGTGATGATAAATGC-3', respectively;
upstream inner primer FIP _ N: 5'-GTGCCGATTAGTTTTGCCAATGCATTGATTTATCTGTCGGT-3', respectively;
the downstream inner primer BIP _ N: 5'-CATTCACCCAATGTATGCTTTCGGGGAATCAATAATCCAACCC-3', respectively;
downstream loop primer LB _ N: 5'-CATTGTTTTAGTGATGGGGGC-3', respectively;
a primer set O':
upstream outer primer F3 — O: 5'-CCTTTGTTATTTTATCCGGC-3', respectively;
downstream outer primer B3 — O: 5'-AGACACAATAAAGCTCATGC-3', respectively;
upstream inner primer FIP _ O: 5'-TGAATGCCATAAGTGCCGATTCTGTCGGTTCAGTTATTGC-3', respectively;
the downstream inner primer BIP _ O: 5'-CGATGTTTGGAGCGTTCATGGCAAGAGTGATGATAAATGC-3', respectively;
downstream loop primer LB _ O: 5'-TGGATTATTGATTCCCTGAAACTCC-3', respectively;
a primer set P':
upstream outer primer F3_ P: 5'-GCTATTGGTTGTGGCGTTTG-3', respectively;
downstream outer primer B3_ P: 5'-AGCAGCAATCGCATCCAA-3', respectively;
upstream inner primer FIP _ P: 5'-TGACCGGAACCCCCATCAGATCGTACCCGCTTTGGTCA-3', respectively;
the downstream inner primer BIP _ P: 5'-CTATCGAGCACTCTGGCTGCTCCTGGCCGCCAATGCATAA-3', respectively;
upstream loop primer LF _ P: 5'-ACCCCCAATGGCGTAAACGTT-3', respectively;
a primer set R':
upstream outer primer F3_ R: 5'-ATCACTTTTGATGGTACGC-3';
downstream outer primer B3_ R: 5'-ACTGTATGCCGTTTGGTT-3', respectively;
an upstream inner primer FIP _ R: 5'-ACTCATCGCCTTCTGAATGGGTGGACCAAGATTGTCATC-3', respectively;
the downstream inner primer BIP _ R: 5'-GCTTAATCCCGTATAGCGCTCCAGCAAACATTAATTCGAC-3', respectively;
downstream loop primer LB _ R: 5'-TGGGCTGCGTTATTCATAGG-3' are provided.
The invention also provides a kit used in the method for detecting the yersinia enterocolitica strain at the constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the yersinia enterocolitica genome. In the kit of the present invention, the primer set capable of amplifying a nucleotide sequence specific to Yersinia enterocolitica genome includes, but is not limited to, a primer sequence including a part of a nucleic acid sequence of 169782 to 171532bp position of genome (GI No. 123440403) or a part of a complementary strand thereof; the primer includes, but is not limited to, any one of the primer set A, the primer set B, the primer set … …, the primer set I, and the like. But not limited to, a primer set having a homology of 50% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set J, primer set K, … …, primer set R, and the like.
In the kit of the present invention, the primer set capable of amplifying the yersinia enterocolitica genome-specific base sequence may include, but is not limited to, a single loop primer; the loop primer serves as an optional component. Preferably, the loop primer is one, including LF or LB. The primer set including the loop primer LF or LB includes, but is not limited to, primer sets A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R', etc. In a specific embodiment, the kit of the invention may comprise 0.4-1.0. mu. mol/L of LF or LB loop primer. In a specific embodiment, the sequences of the primer sets are FIP, BIP, F3, B3, LF or FIP, BIP, F3, B3, LB primers, or primers with 50% or more homology to the aforementioned sequences or their complementary strand sequences.
The kit also comprises Bst DNA polymerase buffer solution, Bst DNA polymerase, dNTP solution and Mg 2+ (MgSO 4 Or MgCl 2 ) And betaine. In a specific embodiment, the enzyme reaction system of the kit comprises 1 XBst DNA polymerase reaction buffer solution and 2-9mmol/L Mg 2+ (MgSO 4 Or MgCl 2 ) 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP and BIP primers, 0.15-0.3 mu mol/L F3 and B3 primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH 8.8), 10mmol/L KCl, 10mmol/L (NH4) 2 SO4，0.1％Triton X-100，2mM MgSO 4 . MgSO in 1 XBst DNA polymerase reaction buffer 4 And magnesium ion Mg in enzyme reaction system 2+ And (6) merging.
The kit of the invention also comprises a positive control template. In a specific embodiment, the positive control template includes, but is not limited to, whole genomic DNA, partial genomic DNA of yersinia enterocolitica, or a vector comprising whole genomic DNA or partial genomic DNA of yersinia enterocolitica.
The kit of the invention further comprises a negative control template, and the negative control template comprises but is not limited to double distilled water.
The kit of the invention also comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein and SYBR GreenI or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn 2+ ]For example, MnCl 2 。
The kit of the invention also comprises double distilled water.
The kit of the invention also comprises a nucleic acid extraction reagent.
The invention also provides a vector, which comprises any one group of primers selected from the group consisting of primer groups A-I, J-R, A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R'. The vector contains a DNA sequence with the specificity of the yersinia enterocolitica, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like, and the application fields of microbial detection and the like. The vector may be, but is not limited to, a plasmid vector (e.g., pBR322, pUC18, pUC19, pBluescript M13, Ti plasmid, etc.), a viral vector (e.g., lambda phage, etc.), and an artificial chromosome vector (e.g., bacterial artificial chromosome BAC, yeast artificial chromosome YAC, etc.). For example, vector pBR322-A containing any one of the primers of primer set A, vector pBR322-J containing any one of the primers of primer set J, and vector pBR322-A 'containing any one of the primers of primer set A' … …. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-J containing any one of the primers of the primer set J, … … a vector lambda phage-A 'containing any one of the primers of the primer set A', and the like.
The invention also provides application of any one primer selected from the primer groups A-I, J-R, A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P 'and R' in constant-temperature detection of Yersinia enterocolitica.
The invention also provides application of the kit in constant-temperature detection of yersinia enterocolitica.
The invention also provides application of the vector in constant-temperature detection of yersinia enterocolitica.
The invention provides a simple, rapid and sensitive method for detecting yersinia enterocolitica, a primer/primer group and a detection reagent/kit for the technical field of food safety detection, and has great significance for food safety in China. The beneficial effects of the invention include: the method for detecting the yersinia enterocolitica has the advantages of strong specificity, high sensitivity, short detection time, simple result judgment, convenient operation, low cost and the like. Compared with the current common detection method, the constant temperature amplification method adopted by the invention can be carried out under the constant temperature condition, only a simple constant temperature device is needed, expensive instruments in PCR experiments are not needed, and the steps of carrying out electrophoresis detection on the amplified products and the like are not needed, so the method is very suitable for being widely applied to various social fields including basic food safety detection departments for popularization and use, and can be fully applied even under the environment with relatively insufficient professional knowledge and skill base of molecular biology. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.
Drawings
FIG. 1 shows the specificity of the constant temperature detection method of Yersinia enterocolitica of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Yersinia enterocolitica detection method of example 8 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1-6 Yersinia enterocolitis isothermal reaction System and detection method
The detection is carried out according to the following steps (1) to (3):
(1) extraction of genomic DNA
Yersinia enterocolitica for detectionThe strain is from China center for the preservation and management of industrial microbial strains, and is numbered CICC 21565. 1mL of the bacterial culture was used to extract genomic DNA and DNA OD using a bacterial nucleic acid extraction kit from Beijing Tiangen bioengineering Co 260 /OD 280 At 1.8, the concentration was 364 ng/. mu.L.
(2) The method comprises the steps of taking genome DNA of yersinia enterocolitica to be detected as a template, respectively adopting self-prepared kits (shown in tables 2 and 3), preparing a reaction system according to conditions in the table 3, and carrying out constant-temperature amplification reaction by taking a specific amplification primer group of the yersinia enterocolitica as a primer. The primers in examples 1 to 6 were primer sets A, A ', I, N, P ', R ', respectively.
(3) The amplification results were confirmed by electrophoresis, turbidity or color development under the conditions shown in Table 3.
As can be seen from Table 3, the detection method and the primer set and the reaction system adopted by the detection method can well amplify specific fragments of Yersinia enterocolitica and obtain detection results. In addition, when the detection is carried out using a detector, the detection effect is also excellent when the reaction time is shortened to 10min (e.g., example 6). Therefore, the invention can be applied to detecting whether the sample contains the yersinia enterocolitica.
According to the method of the above embodiment, the specific fragment of Yersinia enterocolitica can be amplified and the detection result can be obtained by using the primer sets B to H, the primer sets J to M, the primer sets O to R, and the primer sets B ', C ', D ', E ', F ', G ', I ', J ', K ', L ', M ', N ', O ', respectively.
Example 7 Yersinia enterocolitica specific detection
28 strains of Yersinia enterocolitica (1 to 23, 25 to 29 in Table 4 and FIG. 1) were collected, cultured separately from the Yersinia enterocolitica strain (24 in Table 4 and FIG. 1), and 1mL of the bacterial solution was taken, and bacterial DNA was extracted using kit IA, and LAMP amplification (primer set A) and visualization by addition of a color-developing agent were carried out separately with reference to the reaction system and conditions of example 1.
The detection results are shown in Table 4 and FIG. 1, in FIG. 1, 1-23 are Staphylococcus aureus, Staphylococcus aureus Kimbarkii, Staphylococcus epidermidis, Rhodococcus equi, Bacillus cereus, Bacillus mycoides, Listeria monocytogenes, Listeria inoke, Listeria eheli, Salmonella enterica, Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi B, Shigella dysenteriae, Shigella boydii, Shigella flexneri, Escherichia coli (containing Clostridium botulinum type A gene), pathogenic Escherichia coli, Escherichia coli diarrheal, enterotoxigenic Escherichia coli, Escherichia coli enterotoxigenic Escherichia coli, Escherichia enterohemorrhagic Escherichia coli and Cronobacter sakazakii, 25-29 are Pseudotuberculosis, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio and Yersinia enterocolitica, respectively, NTC: negative control, 24: yersinia enterocolitica. In FIG. 1, the product after amplification reaction of Yersinia enterocolitica strain alone appeared in a bright green color, and was a positive result, as shown in tube 24. The products of other Yersinia enterocolitica strains and the negative control amplification reaction are orange, and are negative results, as shown in tubes No. 1-23, No. 25-29 and NTC negative control tube.
As can be seen from the results of FIG. 1 and Table 4, the detection kit and the detection method of the invention have good Yersinia enterocolitis strain specificity, i.e., only Yersinia enterocolitis strain is amplified positively, and other Yersinia enterocolitis strains are negative.
Preparing a detection kit, wherein the primers adopted in the kit are respectively primer groups B-I, primer groups J-R, primer groups A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R', and obtaining the same detection results according to the specific detection method, namely, the products after the amplification reaction of the yersinia enterocolitica strain and the negative control are negative results, and the products after the amplification reaction of the yersinia enterocolitica strain are positive results.
Further, according to the method described in table 1, theoretical analysis was performed on the specificity of each of the primer sets a to I, J to R, a ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R', respectively, and as a result, it was found that, when three mismatches were allowed in each of the primers at most, two primers were simultaneously compared with yersinia enterocolitica at most, indicating that the specificity of each of the primer sets was better.
Example 8 sensitivity detection
DNA of the bacterium CICC21565 was extracted as in example 2, and LAMP amplification (primer set A) and visualization by adding a color-developing agent were carried out in accordance with the method of example 1 in Table 3, respectively, using kit IB, and DNA gradients of 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg and 5fg were added to the reaction system. As shown in fig. 2, 1 to 8 are 50ng, 5ng, 500pg, 50pg, 5pg, 500fg, 50fg and 5fg, respectively, NTC: and (5) negative control. In FIG. 2, the reaction products of 50ng, 5ng, 500pg, 50pg, 5pg and 500fg treatments appeared bright green and as positive results, and the reaction products of 50fg, 5fg treatments and the negative control appeared orange and as negative results. The detection results showed that DNA of 500fg (equivalent to about 100 bacteria) was detected in each reaction tube, and the sensitivity was high.
According to the detection method, other steps and conditions are the same as the above, the primer groups B to I, the primer groups J to R, the primer groups A ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R' are respectively used, and the DNA as low as 5pg to 500fg in each reaction tube can still be detected, so that the detection sensitivity is higher.
Example 9 commonality testing
According to the method described in table 1, theoretical analysis was performed on the versatility of each of the primer sets a to I, J to R, a primer set a ', B', C ', D', E ', F', G ', I', J ', K', L ', M', N ', O', P ', R', and as a result, it was found that the primer region of each primer set perfectly matches three yersinia enterocolitis strains (GI nos. 123440403, 332159624, and 386307442, respectively), and it was theoretically possible to use the detection of the three yersinia enterocolitis strains, indicating that the versatility of each primer set was good.
TABLE 1 analysis of the versatility and specificity of primers in the existing detection methods for Yersinia enterocolitica
Note: a) the sequence between primers F3 and B3 in the patent is Bowtie aligned with three genomes of Yersinia enterocolitica (GI Nos. 123440403, 332159624 and 386307442 respectively) to determine the position of the detection region in the GI No. 123440403 genome. b) The sequences of the detection regions are subjected to Blast comparison in public database resources, and the primer regions are completely matched, so that the universality is good. c) Performing Blast comparison on the detection region sequence in public database resources, wherein the higher the matching degree of the primer region is, the worse the specificity is; if the primers can not be simultaneously compared with the yersinia strain with the non-enterocolitis, the specificity is good.
TABLE 2 types and main components of kit for constant temperature detection of Yersinia enterocolitica
TABLE 3 examples 1-6 reaction conditions and test results in the method for isothermal detection of Yersinia enterocolitica of the present invention
TABLE 4 strains used in the test and the results
Note: a) CGMCC: china general microbiological culture Collection center, CICC: china center for preservation and management of industrial microbial strains, CMCC: china medical bacteria strain preservation and management center. b) +: positive result, -: and (5) negative result.
<110> Shanghai bioinformatics technology research center
Rapid constant-temperature detection method, primer group and kit for yersinia enterocolitica
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Claims (9)
1. A rapid isothermal detection method for yersinia enterocolitica with non-diagnostic purpose, which is characterized by comprising the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) taking the genome DNA as a template, taking a primer group capable of amplifying the specific base sequence of the enterocolitis yersinia genome as a primer, and carrying out constant-temperature amplification reaction in an enzyme reaction system;
(3) determining whether the sample to be detected has yersinia enterocolitica or not by judging whether the reaction result is positive or not;
wherein the genome-specific base sequence of the yersinia enterocolitica is 169782-171532 bp bit sequence of the yersinia enterocolitica genome with the GI number of 123440403;
the primer group capable of amplifying the genome-specific base sequence of the yersinia enterocolitica is selected from the primer group B;
primer set 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);
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3' (SEQ ID NO: 8).
2. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP, 0.8-2.0 mu mol/L FIP _ B and BIP _ B primers, 0.15-0.3 mu mol/L F3_ B and B3_ B primers, 0.16-0.64U/mu L Bst DNA polymerase and 0-1.5mol/L betaine.
3. The method of claim 1, wherein the isothermal amplification reaction is performed by a reaction sequence comprising: incubating for 10-90 min at 60-65 ℃; ② terminating the reaction for 2-20 min at 80 ℃.
4. The primer for the quick constant-temperature detection of the yersinia enterocolitica is characterized by comprising a primer group capable of amplifying a specific base sequence of the yersinia enterocolitica genome, wherein the specific base sequence of the yersinia enterocolitica genome is a part of a nucleic acid sequence with the bit of 169782-171532 bp of the yersinia enterocolitica genome with the GI number of 123440403 or a part of a complementary strand of the nucleic acid sequence;
the primer group capable of amplifying the genome specific base sequence of the yersinia enterocolitica is selected from the primer group B;
primer set 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);
a downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3' (SEQ ID NO: 8).
5. A rapid constant-temperature detection kit for Yersinia enterocolitica is characterized by comprising a primer group capable of amplifying a genome-specific base sequence of Yersinia enterocolitica; the primer group capable of amplifying the genome-specific base sequence of the yersinia enterocolitica is selected from the primer group B;
primer set 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);
the downstream inner primer BIP _ B: 5'-ATTGTGTGGCTATTGACGCGCACTGAACGCAGGTTGATACCA-3' (SEQ ID NO: 8).
6. The kit of claim 5, further comprising a Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg 2+ And one or more of betaine.
7. The kit of claim 5, wherein the enzymatic reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg 2+ 1.0-1.6mmol/L dNTP, 0.8-2.0 μmol/L FIP _ B and BIP _ B primers, 0.15-0.3 μmol/L F3_ B and B3_ B primers, 0.16-0.64U/μ L Bst DNA polymerase, and 0-1.5mol/L betaine.
8. Use of a primer for isothermal detection of yersinia enterocolitica for non-diagnostic purposes, wherein said primer is according to claim 4.
9. Use of a kit according to any one of claims 5 to 7 in isothermal yersinia enterocolitica for non-diagnostic purposes.
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