Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii
阅读说明：本技术 阪崎克罗诺杆菌核酸快速恒温检测方法及试剂盒 (Rapid constant-temperature detection method and kit for nucleic acid of cronobacter sakazakii ) 是由 李雪玲 刘伟 李园园 韦朝春 陆长德 李亦学 贾犇 曹永梅 于 2016-08-30 设计创作，主要内容包括：本发明公开了一种快速恒温检测阪崎克罗诺杆菌的方法、引物组及试剂盒。所述方法为：从待测样品中提取基因组DNA；以所述基因组DNA为模板,以能扩增阪崎克罗诺杆菌特异性序列的引物组为引物,在酶反应体系下进行恒温扩增反应；通过判断反应结果是否为阳性,确定待测样品中是否存在阪崎克罗诺杆菌。本发明检测方法具有高灵敏度和高特异性,检测时间短,结果判定简单,操作便捷,成本低,具广泛应用前景。(The invention discloses a method, a primer group and a kit for rapidly detecting cronobacter sakazakii at constant temperature. The method comprises the following steps: extracting genome DNA from a sample to be detected; performing constant-temperature amplification reaction in an enzyme reaction system by using the genome DNA as a template and a primer group capable of amplifying a specific sequence of Cronobacter sakazakii as a primer; and determining whether the sample to be detected has cronobacter sakazakii 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.)
1. A method for rapidly detecting cronobacter sakazakii at constant temperature 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 Cronobacter sakazakii 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 cronobacter sakazakii or not by judging whether the reaction result is positive or not;
wherein the cronobacter sakazakii genome specific alkali sequence is a sequence of 1077500-1080076 bp bits of the cronobacter sakazakii genome with the GI number of 156932229;
wherein; the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome is selected from any one of the following primer groups M-R;
and (3) primer group M:
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3' (SEQ ID NO: 49);
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3' (SEQ ID NO: 50);
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3' (SEQ ID NO: 51);
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3' (SEQ ID NO: 52);
primer set N:
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3' (SEQ ID NO: 53);
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3' (SEQ ID NO: 54);
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3' (SEQ ID NO: 55);
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3' (SEQ ID NO: 56);
a primer group O:
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3' (SEQ ID NO: 57);
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3' (SEQ ID NO: 58);
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3' (SEQ ID NO: 59);
the downstream inner primer BIP _ O:
5’-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3’(SEQ ID NO：60)；
a primer group P:
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3' (SEQ ID NO: 61);
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3' (SEQ ID NO: 62);
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3' (SEQ ID NO: 63);
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3' (SEQ ID NO: 64);
a primer set Q:
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3' (SEQ ID NO: 65);
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3' (SEQ ID NO: 66);
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3' (SEQ ID NO: 67);
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3' (SEQ ID NO: 68);
a primer set R:
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3' (SEQ ID NO: 69);
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3' (SEQ ID NO: 70);
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3' (SEQ ID NO: 71);
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3' (SEQ ID NO: 72).
2. The method of claim 1, wherein the primer set capable of amplifying the genomic specific base sequence of cronobacter sakazakii further comprises one or more loop primers; the loop primer is LF and/or LB.
3. The method according to claim 2, wherein the primer set capable of amplifying the genomic specific nucleotide sequence of Cronobacter sakazakii is selected from any one of the following primer sets M ', N', O ', P', Q ', R':
a primer set M':
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3', respectively;
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
downstream loop primer LB _ M: 5'-TACGTAGTTCTCCCATGTCCTGGA-3' (SEQ ID NO: 79);
a primer set N':
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3', respectively;
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
upstream loop primer LF _ N: 5'-CGCAAAGCGTCTGGAGTTATTGC-3' (SEQ ID NO: 80);
and/or, the downstream loop primer LB _ N: 5'-TTCAGCCATTCTTCCTGCAGCA-3' (SEQ ID NO: 81);
a primer set O':
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O:
5’-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3’；
upstream loop primer LF _ O: 5'-CTGGAGTTATTGCAATTACTCTCTC-3' (SEQ ID NO: 82);
a primer set P':
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ P: 5'-CATTCAACCTTTACTGCGTGG-3' (SEQ ID NO: 83);
a primer set Q':
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ Q: 5'-GCAGGAAGAATGGCTGAATTTG-3' (SEQ ID NO: 84);
and/or, the downstream loop primer LB _ Q: 5'-CTCTTGTGCCTGCAATAATGC-3' (SEQ ID NO: 85);
a primer set R':
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3', respectively;
downstream loop primer LB _ R: 5'-CAGTAAAGGTTGAATGACAGTGATG-3' (SEQ ID NO. 86).
4. The method of claim 1, wherein in step (2), the enzymatic reaction system comprises: 1 XBstDNA polymerase reaction buffer, 2-9mmol/L Mg2+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, 0-1.5mol/L betaine, including or not including 0.4-1.0. mu. mol/L LF and/or LB primers.
5. The method of claim 1, wherein the isothermal amplification reaction is performed by incubating at ① 60-65 ℃ for 10-90 min and terminating at ② 80 ℃ for 2-20 min.
6. The primer used in the method for isothermal detection of Cronobacter sakazakii according to claim 1, wherein the primer comprises a primer set capable of amplifying a nucleotide sequence specific to the Cronobacter sakazakii genome having a nucleotide sequence of 1077500 to 1080076bp of the Cronobacter sakazakii genome with GI # 156932229 or a complementary strand thereof;
the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome is selected from any one of the following primer groups M-R, and the specific sequence is as follows:
and (3) primer group M:
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3', respectively;
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
primer set N:
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5 '-TTTTGAACAAGCCCGTAATGAC-GGCGTGTTTAAATCGAGA-3';
the downstream inner primer BIP _ N: 5 '-AACCTCAGCTTCTGCCTGTA-ATTAGTTGATGTTAACCGGG-3';
a primer group O:
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O:
5’-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3’；
a primer group P:
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
a primer set Q:
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
a primer set R:
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3' are provided.
7. The primer set of claim 6, wherein the primer set capable of amplifying the genomic specific base sequence of Cronobacter sakazakii further comprises one or more loop primers; the loop primer is LF and/or LB.
8. The primer according to claim 7, wherein the primer set capable of amplifying the genomic specific nucleotide sequence of Cronobacter sakazakii is selected from any one of the following primer sets M ', N', O ', P', Q ', R':
a primer set M':
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3';
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
downstream loop primer LB _ M: 5'-TACGTAGTTCTCCCATGTCCTGGA-3', respectively;
a primer set N':
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3', respectively;
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
upstream loop primer LF _ N: 5'-CGCAAAGCGTCTGGAGTTATTGC-3', respectively;
and/or, the downstream loop primer LB _ N: 5'-TTCAGCCATTCTTCCTGCAGCA-3', respectively;
a primer set O':
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O:
5’-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3’；
upstream loop primer LF _ O: 5'-CTGGAGTTATTGCAATTACTCTCTC-3', respectively;
a primer set P':
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ P: 5'-CATTCAACCTTTACTGCGTGG-3', respectively;
a primer set Q':
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ Q: 5'-GCAGGAAGAATGGCTGAATTTG-3', respectively;
and/or, the downstream loop primer LB _ Q: 5'-CTCTTGTGCCTGCAATAATGC-3', respectively;
a primer set R':
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3', respectively;
downstream loop primer LB _ R: 5'-CAGTAAAGGTTGAATGACAGTGATG-3' are provided.
9. A kit for isothermal detection of Cronobacter sakazakii, comprising the primer according to any one of claims 6 to 8.
10. The kit of claim 9, further comprising a Bst DNA polymerase reaction buffer, Bst DNA polymerase, dNTP solution, Mg2+And one or more of betaine.
11. A kit for isothermal detection of Cronobacter sakazakii, characterized in that an enzyme reaction system of the kit comprises: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+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, with or without 0.4-1.0. mu. mol/L LF and/or LB primers, 0.16-0.64U/. mu.L Bst DNA polymerase, and 0-1.5mol/L betaine.
12. A vector comprising the primer of any one of claims 6 to 8.
13. Use of a primer for isothermal detection of cronobacter sakazakii, wherein the primer is the primer according to any one of claims 6 to 8.
14. Use of the kit according to any one of claims 9 to 11 or the vector according to claim 12 for isothermal detection of cronobacter sakazakii.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method, primers and a kit for rapidly detecting cronobacter sakazakii at a constant temperature.
Background
Sakazakii (Cronobacter sakazakii) is a gram-negative, peritrichous, motile, spore-free facultative anaerobic bacterium known as atypical, flavochrome-producing enterobacter cloacae before 1980, and renamed as enterobacter sakazakii after 1980, and reclassified in 2008, classified as crohn's bacillus of enterobacteriaceae. The cronobacter sakazakii is widely existed in the natural world, is an important conditioned pathogen which harms the health of infants through the formula powder, can cause serious clinical symptoms of the infants, such as cerebral abscess, meningitis, necrotizing enterocolitis, systemic septicemia and the like, and has the lethality rate of 40-80%. Both newborn infants and premature infants are at risk of infection with cronobacter sakazakii strains by consumption of infant formula contaminated with cronobacter sakazakii. Compared with other food-borne pathogenic microorganisms, the cronobacter sakazakii has a low infection rate but has a high mortality rate for special people, and the host and the propagation model of the cronobacter sakazakii are not clear yet. Therefore, it is particularly important to prevent and detect the bacteria.
At present, the detection method for cronobacter sakazakii at home and abroad still uses a conventional culture method as a standard, but the detection period is longer (up to 5-7 days), the operation is relatively complex, the detection efficiency is lower, and 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. Researchers developed detection means of PCR and fluorescence PCR technologies successively, and the detection means is also brought into the industry standard of 'detection method of Cronobacter sakazakii in milk powder' in China, but the two methods need special detection instruments, and therefore, the method is not suitable for being widely applied to basic detection departments, particularly real-time field detection in enterprise production lines.
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, and upstream and downstream inner primers FIP and BIP, wherein FIP is composed of F1C and F2, and BIP is composed 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, and generates a visible reaction by-product, white magnesium pyrophosphate precipitate (see Notomi T, OkayamaH, Masubuchi H, Yonekawa T, Watanabe K, Nuino N, Hase T. loop-mediated isothermal amplification reaction (2000, J8512; 63). 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 of the species to be tested. Taking the invention patents ZL201010614700.1 and CN 101319249B as examples, the OmpA gene and the 18S rDNA sequence, which are specific genes of Cronobacter sakazakii and are reported in the literature, are respectively used for detecting the Cronobacter sakazakii by adopting LAMP technology. However, the so-called "recognized specific gene" is often based on the knowledge of hysteresis and is not necessarily updated based on the ever-increasing genome data of microorganisms, so that a primer obtained based on the sequence of the target gene is not necessarily able to ensure its specificity in practical use. The present invention, as shown in Table 1, shows the problems of insufficient primer specificity and unsuitability of versatility in the prior art. That is, the cronobacter sakazakii specific sequence used in the prior art method is not actually specific to cronobacter sakazakii, that is, it is possible that cronobacter sakazakii other than cronobacter sakazakii is erroneously identified as cronobacter sakazakii. Similar problems exist with respect to validation of versatility. Therefore, a cronobacter sakazakii detection method capable of ensuring specificity and universality is urgently needed in the industry, and meanwhile, the requirements of the primary detection department on rapidness and convenience are met, and real-time on-site detection can be conveniently carried out 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 cronobacter sakazakii, and forms a high-sensitivity and high-specificity detection kit on the basis. The invention designs a Cronobacter sakazakii LAMP primer based on microbial genome data resources (
The invention provides a method for rapidly detecting Cronobacter sakazakii strains, which comprises the following steps:
(1) extracting genome DNA from a sample to be detected;
(2) carrying out constant-temperature amplification reaction in an enzyme reaction system by taking the genome DNA as a template and a primer group capable of amplifying the specific base sequence of the Cronobacter sakazakii genome as a primer;
(3) and determining whether the sample to be detected has cronobacter sakazakii or not by judging whether the reaction result is positive or not.
The method for detecting the cronobacter sakazakii strain at constant temperature extracts genome DNA from a sample to be detected, takes the genome DNA as a template and takes a cronobacter sakazakii specific amplification primer group as a primer to carry out constant temperature amplification reaction, and then determines whether the cronobacter sakazakii exists in the sample to be detected 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 alkali sequence of the cronobacter sakazakii is the sequence of 1077500-1080076 bp bits of the cronobacter sakazakii with the GI number of 156932229.
In the present invention, the primer set capable of amplifying the nucleotide sequence specific to the Cronobacter sakazakii genome is a part of the nucleotide sequence of 1077500 to 1080076bp of the genome (GI No. 156932229) or a part of the complementary strand thereof. Wherein the sakazakii genome-specific nucleotide sequence is a nucleotide sequence unique to the sakazakii genome, and is not contained in the genome of another microorganism.
Wherein the primer set capable of amplifying the specific nucleotide sequence of the Cronobacter sakazakii genome includes, but is not limited to, any one selected from the following primer sets A to L, or any one selected from the primer sets having a homology of 45% or more with a single sequence in the sequence of the primer set or the complementary strand sequence thereof.
Primer set a:
upstream outer primer F3_ a: 5'-GGATTAGGAAATATACGACGC-3' (SEQ ID NO: 1);
downstream outer primer B3_ a: 5'-AGCATATATGATACGGGGAA-3' (SEQ ID NO: 2);
upstream inner primer FIP _ A: 5'-AGCAAAACCCATTGGTACCTTATACTGTCTATGGGCACAA-3' (SEQ ID NO: 3);
the downstream inner primer BIP _ A: 5'-TGAATATGATCCCCCGGCTTCTGGGAGGAAATATCGCTAA-3' (SEQ ID NO: 4);
primer set B:
upstream outer primer F3_ B: 5'-GTTCGATCTCCAGCTTACGAAG-3' (SEQ ID NO: 5);
downstream outer primer B3_ B: 5'-GACATGAGTATCCACCAGGC-3' (SEQ ID NO: 6);
upstream inner primer FIP _ B: 5'-GCGAGAGGTAACGAACGCAGTGGCGAGATTTCATATTGCCGC-3' (SEQ ID NO: 7);
the downstream inner primer BIP _ B: 5'-AGTTGCTGGTGAGCTTCGGCAGAAGTACAGGCCAGGGTAA-3' (SEQ ID NO: 8);
primer set C:
upstream outer primer F3_ C: 5'-GGATACATCCCATTGGCT-3' (SEQ ID NO: 9);
downstream outer primer B3 — C: 5'-CGTCTGGAGTTATTGCAATT-3' (SEQ ID NO: 10);
upstream inner primer FIP _ C: 5'-AAGCAAGCAGAATACTCCCAGCTGCCAGGTCAATATTTGC-3' (SEQ ID NO: 11);
the downstream inner primer BIP _ C: 5'-CATCAACTTTTGTATCATGTGCCTGTCTCGATTTAAACACGCC-3' (SEQ ID NO: 12);
primer set D:
upstream outer primer F3_ D: 5'-ATCATGTGCCTGAAGTGA-3' (SEQ ID NO: 13);
downstream outer primer B3_ D: 5'-TTAACCGGGCAATGATTG-3' (SEQ ID NO: 14);
upstream inner primer FIP _ D: 5'-GTAATGACCTTGACGCAAAGCGTTGCTTCTATTGGCGTG-3' (SEQ ID NO: 15);
the downstream inner primer BIP _ D:
5’-GTTCAAAATTTAGTTCCTGAGAGGCGAAGAATGGCTGAATTTGAG-3’(SEQ ID NO：16)；
primer set E:
upstream outer primer F3_ E: 5'-TGAAGTGATTCGGTTGCT-3' (SEQ ID NO: 17);
downstream outer primer B3_ E: 5'-GCGAATCTCTTAATCGATCA-3' (SEQ ID NO: 18);
upstream inner primer FIP _ E: 5'-GAACAAGCCCGTAATGACCTTCGAGAGAGAGTAATTGCA-3' (SEQ ID NO: 19);
the downstream inner primer BIP _ E: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3' (SEQ ID NO: 20);
a primer set F:
upstream outer primer F3 — F: 5'-CTGCCTGTACAATCTCAAA-3' (SEQ ID NO: 21);
downstream outer primer B3 — F: 5'-GAGCAAATTAACCGGCTTAA-3' (SEQ ID NO: 22);
upstream inner primer FIP _ F: 5'-GCTAAAAATTGCTCGCGAATCTCCATTCTTCCTGCAGCAAT-3' (SEQ ID NO: 23);
the downstream inner primer BIP _ F: 5'-TGCTCTTGTGCCTGCAATTTTCACAAACTGTCACCG-3' (SEQ ID NO: 24);
primer set G:
upstream outer primer F3_ G: 5'-AATTTGCTCACTCAGTTGTG-3' (SEQ ID NO: 25);
downstream outer primer B3_ G: 5'-ATGACCGTTATCGTCAGG-3' (SEQ ID NO: 26);
upstream inner primer FIP _ G: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3' (SEQ ID NO: 27);
the downstream inner primer BIP _ G:
5’-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3’(SEQ ID NO：28)；
a primer set H:
upstream outer primer F3 — H: 5'-AAGGCTTACTCTTGAACCA-3' (SEQ ID NO: 29);
downstream outer primer B3 — H: 5'-TGGGACTCCGAAACAATC-3' (SEQ ID NO: 30);
upstream inner primer FIP _ H:
5’-CACATATAACCAGGATGACCGTTATTAAGGTGTCAGGATAGTGG-3’(SEQ ID NO：31)；
the downstream inner primer BIP _ H: 5'-GCTAAGCGTCAATTTCGGGTGCGCTTATTTGAACCGTAT-3' (SEQ ID NO: 32);
primer set I:
upstream outer primer F3_ I: 5'-GGCAGAAACAGCCTGTTCA-3' (SEQ ID NO: 33);
downstream outer primer B3 — I: 5'-TGGCTGGGGAACTTTCTCAT-3' (SEQ ID NO: 34);
upstream inner primer FIP _ I: 5'-CCTGTCCAGGCATTACGTGATGAGCATCTACACTGACAGGCAC-3' (SEQ ID NO: 35);
the downstream inner primer BIP _ I: 5'-TAAACGCGCCGCAGAGACTGTTATGCTGGGTGTTGGCG-3' (SEQ ID NO: 36);
primer set J:
upstream outer primer F3_ J: 5'-ATAAGGCCCATAAGTGCG-3' (SEQ ID NO: 37);
downstream outer primer B3_ J: 5'-AGGTATGGAAAAACAGTCAC-3' (SEQ ID NO: 38);
upstream inner primer FIP _ J: 5'-TTGGCGTCCGTATAGCGTTACCAGTAAGAACGGTGGAT-3' (SEQ ID NO: 39);
the downstream inner primer BIP _ J: 5'-CATCACCATAACATTCATAACACCGTTTCTGGTCTTGGAGGAAT-3' (SEQ ID NO: 40);
primer set K:
upstream outer primer F3_ K: 5'-GTCCCACTTTGAGATGCA-3' (SEQ ID NO: 41);
downstream outer primer B3_ K: 5'-GACTATTCAACTGCCTGTT-3' (SEQ ID NO: 42);
upstream inner primer FIP _ K: 5'-GCCCCTTTAGTCTCAGCAAATTTATCAGTGCTCCCTATCG-3' (SEQ ID NO: 43);
the downstream inner primer BIP _ K: 5'-CTGCAATTGTTTCAGGAATCAGTGGAAACGAGACAATTTCGGT-3' (SEQ ID NO: 44);
a primer set L:
upstream outer primer F3_ L: 5'-TCATTGCCTGTAATTGCG-3' (SEQ ID NO: 45);
downstream outer primer B3_ L: 5'-TGAAAGATCTCATGCCAAC-3' (SEQ ID NO: 46);
upstream inner primer FIP _ L: 5'-GTGCATTTCTGGCTCTCATTGTTTAGTAACGCAACGACG-3' (SEQ ID NO: 47);
the downstream inner primer BIP _ L:
5’-CTCGTAAAATGTGTAAGCAAGATGGGGCTAAACGCAATACATTCA-3’(SEQ ID NO：48)。
in the present invention, the primer set capable of amplifying the specific nucleotide sequence of the cronobacter sakazakii genome may further include a primer set having a homology of 45% or more with a single sequence in each of the aforementioned primer set sequences or the complementary strand sequences thereof, and the primer set includes, but is not limited to, any one of the following primer sets M to R:
and (3) primer group M:
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3' (SEQ ID NO: 49);
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3' (SEQ ID NO: 50) (50% homology to the complementary strand 5'-AGCCAATGGGATGTATCC-3' of primer F3_ C);
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3' (SEQ ID NO: 51);
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3' (SEQ ID NO: 52);
primer set N:
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3' (SEQ ID NO: 53) (homology 50% to primer F3_ D5 '-ATCATGTGCCTGAAGTGA-3');
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3' (SEQ ID NO: 54);
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3' (SEQ ID NO: 55);
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3' (SEQ ID NO: 56);
a primer group O:
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3' (SEQ ID NO: 57) (homology 50% to primer F3_ E5 '-TGAAGTGATTCGGTTGCT-3');
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3' (SEQ ID NO: 58);
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3' (SEQ ID NO: 59);
the downstream inner primer BIP _ O: 5'-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3' (SEQ ID NO: 60);
a primer group P:
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3' (SEQ ID NO: 61) (homology 45% to the complementary strand 5'-TTAAGCCGGTTAATTTGCTC-3' of primer B3_ F);
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3' (SEQ ID NO: 62);
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3' (SEQ ID NO: 63);
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3' (SEQ ID NO: 64);
a primer set Q:
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3' (SEQ ID NO: 65);
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3' (SEQ ID NO: 66) (50% homology to the complementary strand 5'-CACAACTGAGTGAGCAAATT-3' of primer F3_ G);
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3' (SEQ ID NO: 67);
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3' (SEQ ID NO: 68);
a primer set R:
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3' (SEQ ID NO: 69);
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3' (SEQ ID NO: 70) (47% homology to the complementary strand 5'-TGGTTCAAGAGTAAGCCTT-3' of primer F3_ H);
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3' (SEQ ID NO: 71);
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3' (SEQ ID NO: 72).
In the method of the present invention, the primer set capable of amplifying the genomic specific nucleotide sequence of cronobacter sakazakii may or may not contain a loop primer. The loop primer may be one or more, including primers LF and/or LB. The primer group capable of amplifying the specific base sequence of the sakazakii cronobacter genome is selected from any one of the following primer groups A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ' and R '; or any one selected from the group consisting of primer groups having a homology of 45% or more with respect to a single sequence in the sequences of the primer groups A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ', R ' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GGATTAGGAAATATACGACGC-3', respectively;
downstream outer primer B3_ a: 5'-AGCATATATGATACGGGGAA-3', respectively;
upstream inner primer FIP _ A: 5'-AGCAAAACCCATTGGTACCTTATACTGTCTATGGGCACAA-3', respectively;
the downstream inner primer BIP _ A: 5'-TGAATATGATCCCCCGGCTTCTGGGAGGAAATATCGCTAA-3', respectively;
downstream loop primer LB _ a: 5'-ATGTCTTTCCCTGCATTTTCCA-3' (SEQ ID NO: 73);
a primer set D':
upstream outer primer F3_ D: 5'-ATCATGTGCCTGAAGTGA-3', respectively;
downstream outer primer B3_ D: 5'-TTAACCGGGCAATGATTG-3', respectively;
upstream inner primer FIP _ D: 5'-GTAATGACCTTGACGCAAAGCGTTGCTTCTATTGGCGTG-3', respectively;
the downstream inner primer BIP _ D:
5’-GTTCAAAATTTAGTTCCTGAGAGGCGAAGAATGGCTGAATTTGAG-3’；
upstream loop primer LF _ D: 5'-GAGTTATTGCAATTACTCTCTCTCG-3' (SEQ ID NO: 74);
and/or, the downstream loop primer LB _ D: 5'-CTCAGCTTCTGCCTGTACAA-3' (SEQ ID NO: 75);
a primer set E':
upstream outer primer F3_ E: 5'-TGAAGTGATTCGGTTGCT-3', respectively;
downstream outer primer B3_ E: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ E: 5'-GAACAAGCCCGTAATGACCTTCGAGAGAGAGTAATTGCA-3', respectively;
the downstream inner primer BIP _ E: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
downstream loop primer LB _ E: 5'-TCTCAAATTCAGCCATTCTTCCTG-3' (SEQ ID NO: 76);
a primer set F':
upstream outer primer F3 — F: 5'-CTGCCTGTACAATCTCAAA-3', respectively;
downstream outer primer B3 — F: 5'-GAGCAAATTAACCGGCTTAA-3', respectively;
upstream inner primer FIP _ F: 5'-GCTAAAAATTGCTCGCGAATCTCCATTCTTCCTGCAGCAAT-3', respectively;
the downstream inner primer BIP _ F: 5'-TGCTCTTGTGCCTGCAATTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ F: 5'-AGTTGATGTTAACCGGGCAAT-3' (SEQ ID NO: 77);
a primer set G':
upstream outer primer F3_ G: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ G: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ G: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ G: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ G: 5'-CATTCAACCTTTACTGCGTGG-3' (SEQ ID NO: 78);
a primer set M':
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3';
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
downstream loop primer LB _ M: 5'-TACGTAGTTCTCCCATGTCCTGGA-3' (SEQ ID NO: 79);
a primer set N':
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3', respectively;
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
upstream loop primer LF _ N: 5'-CGCAAAGCGTCTGGAGTTATTGC-3' (SEQ ID NO: 80);
and/or, the downstream loop primer LB _ N: 5'-TTCAGCCATTCTTCCTGCAGCA-3' (SEQ ID NO: 81);
a primer set O':
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O: 5'-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3', respectively;
upstream loop primer LF _ O: 5'-CTGGAGTTATTGCAATTACTCTCTC-3' (SEQ ID NO: 82);
a primer set P':
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ P: 5'-CATTCAACCTTTACTGCGTGG-3' (SEQ ID NO: 83);
a primer set Q':
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ Q: 5'-GCAGGAAGAATGGCTGAATTTG-3' (SEQ ID NO: 84);
and/or, the downstream loop primer LB _ Q: 5'-CTCTTGTGCCTGCAATAATGC-3' (SEQ ID NO: 85);
a primer set R':
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3', respectively;
downstream loop primer LB _ R: 5'-CAGTAAAGGTTGAATGACAGTGATG-3' (SEQ ID NO. 86).
In specific embodiments, for example, the primer set D ', N ' or Q ' may comprise only one forward loop primer, only one downstream loop primer, or both an upstream loop primer and a downstream loop primer.
In the method of the present invention, in a specific embodiment (including loop primer), the isothermal amplification enzyme reaction systemComprises the following steps: 1 XBst DNA polymerase reaction buffer, 2-9mmol/L Mg2+(MgSO4Or MgCl2) 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 and/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 Mg2+(MgSO4Or MgCl2) 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 the improvement of the reaction efficiency. For example, 1 XBst DNA polymerase reaction buffer can be 1 × Thermopol reaction buffer containing 20mmol/L Tris-HCl (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+And (6) merging.
In the method, the reaction procedure of the constant-temperature amplification reaction is incubation at ① 60-65 ℃ for 10-90 min, preferably 20-90 min, preferably 10-60 min, and termination reaction at ② 80 ℃ for 2-20 min.
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 chart shows a characteristic step-shaped strip, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if the electrophoretogram does not present a characteristic ladder-shaped strip, the sample to be detected is negative to cronobacter sakazakii. The turbidity detection is to detect turbidity by naked eye observation or a turbidity meter, and if the detection tube is turbid, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if no turbidity is found, the sample to be tested is negative to cronobacter sakazakii. Or the reaction tube bottom can be visually observed whether the precipitate exists after the centrifugation, if the precipitate exists at the reaction tube bottom, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii; if the reaction tube bottom is not precipitated, the sample to be tested is negative to cronobacter sakazakii.
The color development detection is to add color development reagent, including but not limited to calcein (50 μ M) or SYBRGreen I (30-50X), or hydroxynaphthol blue (i.e. HNB, 120-. When calcein or SYBR Green I is used as a color developing agent, if the color is orange after reaction, the sample to be detected is negative to cronobacter sakazakii; if the color after the reaction is green, the sample to be detected is positive to the cronobacter sakazakii and contains the cronobacter sakazakii. 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 cronobacter sakazakii; if the color after the reaction is sky blue, the sample to be detected is positive to the cronobacter sakazakii. The chromogenic detection can be used for detecting the reaction result in real time or at an end point through a detection instrument besides observing the reaction result through naked eyes, and by reasonably setting a threshold value of 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 sakazakii; 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 to the cronobacter sakazakii. 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 GenieII 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, 50 μ M calcein is added into enzyme reaction system, and 0.6-1mM [ Mn ] is added2+]For example, 0.6-1mM MnCl2。
The invention also provides a primer used in the method for detecting the cronobacter sakazakii strain at constant temperature. The primer comprises a primer group capable of amplifying specific base sequences of a cronobacter sakazakii genome, and the primer comprises but is not limited to a part of a nucleic acid sequence with the GI number of 156932229 at the 1077500-1080076 bp position of the cronobacter sakazakii genome or a part of a complementary strand thereof.
Wherein the primer group capable of amplifying the specific nucleotide sequence of the Cronobacter sakazakii genome is selected from any one of the following primer groups, or is selected from any one of the primer groups having a homology of 45% or more with a single sequence in the sequence of each primer group or the sequence of the complementary strand thereof. Wherein the primer set includes, but is not limited to, any one of the following primer sets A to L. The primer set having a homology of 45% 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 M to R.
Primer set a:
upstream outer primer F3_ a: 5'-GGATTAGGAAATATACGACGC-3', respectively;
downstream outer primer B3_ a: 5'-AGCATATATGATACGGGGAA-3', respectively;
upstream inner primer FIP _ A: 5'-AGCAAAACCCATTGGTACCTTATACTGTCTATGGGCACAA-3', respectively;
the downstream inner primer BIP _ A: 5'-TGAATATGATCCCCCGGCTTCTGGGAGGAAATATCGCTAA-3', respectively;
primer set B:
upstream outer primer F3_ B: 5'-GTTCGATCTCCAGCTTACGAAG-3', respectively;
downstream outer primer B3_ B: 5'-GACATGAGTATCCACCAGGC-3', respectively;
upstream inner primer FIP _ B: 5'-GCGAGAGGTAACGAACGCAGTGGCGAGATTTCATATTGCCGC-3', respectively;
the downstream inner primer BIP _ B: 5'-AGTTGCTGGTGAGCTTCGGCAGAAGTACAGGCCAGGGTAA-3', respectively;
primer set C:
upstream outer primer F3_ C: 5'-GGATACATCCCATTGGCT-3', respectively;
downstream outer primer B3 — C: 5'-CGTCTGGAGTTATTGCAATT-3', respectively;
upstream inner primer FIP _ C: 5'-AAGCAAGCAGAATACTCCCAGCTGCCAGGTCAATATTTGC-3', respectively;
the downstream inner primer BIP _ C: 5'-CATCAACTTTTGTATCATGTGCCTGTCTCGATTTAAACACGCC-3', respectively;
primer set D:
upstream outer primer F3_ D: 5'-ATCATGTGCCTGAAGTGA-3', respectively;
downstream outer primer B3_ D: 5'-TTAACCGGGCAATGATTG-3', respectively;
upstream inner primer FIP _ D: 5'-GTAATGACCTTGACGCAAAGCGTTGCTTCTATTGGCGTG-3', respectively;
the downstream inner primer BIP _ D: 5'-GTTCAAAATTTAGTTCCTGAGAGGCGAAGAATGGCTGAATTTGAG-3', respectively;
primer set E:
upstream outer primer F3_ E: 5'-TGAAGTGATTCGGTTGCT-3', respectively;
downstream outer primer B3_ E: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ E: 5'-GAACAAGCCCGTAATGACCTTCGAGAGAGAGTAATTGCA-3', respectively;
the downstream inner primer BIP _ E: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
a primer set F:
upstream outer primer F3 — F: 5'-CTGCCTGTACAATCTCAAA-3', respectively;
downstream outer primer B3 — F: 5'-GAGCAAATTAACCGGCTTAA-3', respectively;
upstream inner primer FIP _ F: 5'-GCTAAAAATTGCTCGCGAATCTCCATTCTTCCTGCAGCAAT-3', respectively;
the downstream inner primer BIP _ F: 5'-TGCTCTTGTGCCTGCAATTTTCACAAACTGTCACCG-3', respectively;
primer set G:
upstream outer primer F3_ G: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ G: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ G: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ G: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
a primer set H:
upstream outer primer F3 — H: 5'-AAGGCTTACTCTTGAACCA-3', respectively;
downstream outer primer B3 — H: 5'-TGGGACTCCGAAACAATC-3', respectively;
upstream inner primer FIP _ H: 5'-CACATATAACCAGGATGACCGTTATTAAGGTGTCAGGATAGTGG-3', respectively;
the downstream inner primer BIP _ H: 5'-GCTAAGCGTCAATTTCGGGTGCGCTTATTTGAACCGTAT-3', respectively;
primer set I:
upstream outer primer F3_ I: 5'-GGCAGAAACAGCCTGTTCA-3', respectively;
downstream outer primer B3 — I: 5'-TGGCTGGGGAACTTTCTCAT-3', respectively;
upstream inner primer FIP _ I: 5'-CCTGTCCAGGCATTACGTGATGAGCATCTACACTGACAGGCAC-3', respectively;
the downstream inner primer BIP _ I: 5'-TAAACGCGCCGCAGAGACTGTTATGCTGGGTGTTGGCG-3', respectively;
primer set J:
upstream outer primer F3_ J: 5'-ATAAGGCCCATAAGTGCG-3', respectively;
downstream outer primer B3_ J: 5'-AGGTATGGAAAAACAGTCAC-3', respectively;
upstream inner primer FIP _ J: 5'-TTGGCGTCCGTATAGCGTTACCAGTAAGAACGGTGGAT-3', respectively;
the downstream inner primer BIP _ J: 5'-CATCACCATAACATTCATAACACCGTTTCTGGTCTTGGAGGAAT-3', respectively;
primer set K:
upstream outer primer F3_ K: 5'-GTCCCACTTTGAGATGCA-3', respectively;
downstream outer primer B3_ K: 5'-GACTATTCAACTGCCTGTT-3', respectively;
upstream inner primer FIP _ K: 5'-GCCCCTTTAGTCTCAGCAAATTTATCAGTGCTCCCTATCG-3', respectively;
the downstream inner primer BIP _ K:
5’-CTGCAATTGTTTCAGGAATCAGTGGAAACGAGACAATTTCGGT-3’；
a primer set L:
upstream outer primer F3_ L: 5'-TCATTGCCTGTAATTGCG-3', respectively;
downstream outer primer B3_ L: 5'-TGAAAGATCTCATGCCAAC-3', respectively;
upstream inner primer FIP _ L: 5'-GTGCATTTCTGGCTCTCATTGTTTAGTAACGCAACGACG-3', respectively;
the downstream inner primer BIP _ L: 5'-CTCGTAAAATGTGTAAGCAAGATGGGGCTAAACGCAATACATTCA-3', respectively;
and (3) primer group M:
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3', respectively;
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
primer set N:
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3', respectively;
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
a primer group O:
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O: 5'-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3', respectively;
a primer group P:
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
a primer set Q:
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
a primer set R:
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3' are provided.
In the primer used in the method for detecting cronobacter sakazakii at constant temperature, the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome may or may not comprise one or more loop primers; the loop primer is LF and/or LB. The primer group capable of amplifying the specific base sequence of the sakazakii cronobacter genome is selected from any one of the following primer groups A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ' and R '; or any one selected from the group consisting of primer groups having a homology of 45% or more with respect to a single sequence in the sequences of the primer groups A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ', R ' or the complementary strand sequences thereof:
primer set a':
upstream outer primer F3_ a: 5'-GGATTAGGAAATATACGACGC-3', respectively;
downstream outer primer B3_ a: 5'-AGCATATATGATACGGGGAA-3', respectively;
upstream inner primer FIP _ A: 5'-AGCAAAACCCATTGGTACCTTATACTGTCTATGGGCACAA-3', respectively;
the downstream inner primer BIP _ A: 5'-TGAATATGATCCCCCGGCTTCTGGGAGGAAATATCGCTAA-3', respectively;
downstream loop primer LB _ a: 5'-ATGTCTTTCCCTGCATTTTCCA-3', respectively;
a primer set D':
upstream outer primer F3_ D: 5'-ATCATGTGCCTGAAGTGA-3', respectively;
downstream outer primer B3_ D: 5'-TTAACCGGGCAATGATTG-3', respectively;
upstream inner primer FIP _ D: 5'-GTAATGACCTTGACGCAAAGCGTTGCTTCTATTGGCGTG-3', respectively;
the downstream inner primer BIP _ D: 5'-GTTCAAAATTTAGTTCCTGAGAGGCGAAGAATGGCTGAATTTGAG-3', respectively;
upstream loop primer LF _ D: 5'-GAGTTATTGCAATTACTCTCTCTCG-3', respectively;
and/or, the downstream loop primer LB _ D: 5'-CTCAGCTTCTGCCTGTACAA-3', respectively;
a primer set E':
upstream outer primer F3_ E: 5'-TGAAGTGATTCGGTTGCT-3', respectively;
downstream outer primer B3_ E: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ E: 5'-GAACAAGCCCGTAATGACCTTCGAGAGAGAGTAATTGCA-3', respectively;
the downstream inner primer BIP _ E: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
downstream loop primer LB _ E: 5'-TCTCAAATTCAGCCATTCTTCCTG-3', respectively;
a primer set F':
upstream outer primer F3 — F: 5'-CTGCCTGTACAATCTCAAA-3', respectively;
downstream outer primer B3 — F: 5'-GAGCAAATTAACCGGCTTAA-3', respectively;
upstream inner primer FIP _ F: 5'-GCTAAAAATTGCTCGCGAATCTCCATTCTTCCTGCAGCAAT-3', respectively;
the downstream inner primer BIP _ F: 5'-TGCTCTTGTGCCTGCAATTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ F: 5'-AGTTGATGTTAACCGGGCAAT-3', respectively;
a primer set G':
upstream outer primer F3_ G: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ G: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ G: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ G: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ G: 5'-CATTCAACCTTTACTGCGTGG-3', respectively;
a primer set M':
upstream outer primer F3_ M: 5'-GCACTGCGTTCGTTACCT-3', respectively;
downstream outer primer B3_ M: 5'-GATGTATCCCTTGTTGGTGGAG-3', respectively;
upstream inner primer FIP _ M: 5'-GTACAGGCCAGGGTAAATGTACGTCCAGTTGCTGGTGAGCTT-3', respectively;
the downstream inner primer BIP _ M: 5'-TGACCGGTATTTCGACCTGTATGCCAAGTGCGCAGACGAGAT-3', respectively;
downstream loop primer LB _ M: 5'-TACGTAGTTCTCCCATGTCCTGGA-3', respectively;
a primer set N':
upstream outer primer F3 — N: 5'-CTGAAGTGATTCGGTTGC-3', respectively;
downstream outer primer B3 — N: 5'-GCGAATCTCTTAATCGATCA-3', respectively;
upstream inner primer FIP _ N: 5'-TTTTGAACAAGCCCGTAATGACGGCGTGTTTAAATCGAGA-3', respectively;
the downstream inner primer BIP _ N: 5'-AACCTCAGCTTCTGCCTGTAATTAGTTGATGTTAACCGGG-3', respectively;
upstream loop primer LF _ N: 5'-CGCAAAGCGTCTGGAGTTATTGC-3', respectively;
and/or, the downstream loop primer LB _ N: 5'-TTCAGCCATTCTTCCTGCAGCA-3', respectively;
a primer set O':
upstream outer primer F3 — O: 5'-TATCATGTGCCTGAAGTGAT-3', respectively;
downstream outer primer B3 — O: 5'-GTTAACCGGGCAATGATT-3', respectively;
upstream inner primer FIP _ O: 5'-GTAATGACCTTGACGCAAAGCCTATTGGCGTGTTTAAATCG-3', respectively;
the downstream inner primer BIP _ O: 5'-CAAAATTTAGTTCCTGAGAGGCAACGAAGAATGGCTGAATTTGAG-3', respectively;
upstream loop primer LF _ O: 5'-CTGGAGTTATTGCAATTACTCTCTC-3', respectively;
a primer set P':
upstream outer primer F3_ P: 5'-AATTTGCTCACTCAGTTGTG-3', respectively;
downstream outer primer B3_ P: 5'-ATGACCGTTATCGTCAGG-3', respectively;
upstream inner primer FIP _ P: 5'-GCTTTTCCACAAACGATGGTGGTTGCAATATTTTTACCGGC-3', respectively;
the downstream inner primer BIP _ P: 5'-TATAGTTCCATCCAAGGCTTACTCGTGTCACCAAAAACCACTAT-3', respectively;
upstream loop primer LF _ P: 5'-CATTCAACCTTTACTGCGTGG-3', respectively;
a primer set Q':
upstream outer primer F3_ Q: 5'-TTTAGTTCCTGAGAGGCAA-3', respectively;
downstream outer primer B3_ Q: 5'-TGAGCAAATTAACCGGCT-3', respectively;
upstream inner primer FIP _ Q: 5'-GATGTTAACCGGGCAATGATTGGCTTCTGCCTGTACAATC-3', respectively;
the downstream inner primer BIP _ Q: 5'-GATTCGCGAGCAATTTTTAGCTTTTCACAAACTGTCACCG-3', respectively;
upstream loop primer LF _ Q: 5'-GCAGGAAGAATGGCTGAATTTG-3', respectively;
and/or, the downstream loop primer LB _ Q: 5'-CTCTTGTGCCTGCAATAATGC-3', respectively;
a primer set R':
upstream outer primer F3_ R: 5'-GCAATTTTTAGCTGCTCTTG-3', respectively;
downstream outer primer B3_ R: 5'-GTAAGCCTTGGATGGAACT-3', respectively;
upstream inner primer FIP _ R: 5'-GTGAGCAAATTAACCGGCTTAAAAGAAGTAATGATCCCGGT-3', respectively;
the downstream inner primer BIP _ R: 5'-GCAATATTTTTACCGGCACCACATATGGCTGGCTTTTCCA-3', respectively;
downstream loop primer LB _ R: 5'-CAGTAAAGGTTGAATGACAGTGATG-3' are provided.
In a specific embodiment, the primer set D ', N ' or Q ' may comprise only one forward loop primer, only one downstream loop primer, or both a forward loop primer and a downstream loop primer. In a specific embodiment, the primers are respectively FIP, BIP, F3, B3, LF and LB primers or primers with 45% or more homology with the above primer sequence or single primer in the complementary strand sequence.
The invention also provides a kit used in the method for detecting the cronobacter sakazakii strain at constant temperature, which comprises the primer group capable of amplifying the specific base sequence of the cronobacter sakazakii genome. In the kit of the present invention, the primer set capable of amplifying the nucleotide sequence specific to the Cronobacter sakazakii genome includes, but is not limited to, a primer sequence including a part of the nucleic acid sequence at the 1077500 to 1080076bp position of the genome (GI No: 156932229) or a part of the 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 K, the primer set L, and the like. But not limited to, a primer set having a homology of 45% or more with the aforementioned primer sequence or a single sequence in the complementary strand sequence thereof; including but not limited to primer set M, primer set N, … …, primer set Q, primer set R, and the like.
In the kit of the present invention, the primer set capable of amplifying the sakazakii genome-specific base sequence may or may not comprise one or more loop primers; the loop primer serves as an optional component. The loop primer is LF and/or LB. Primer sets comprising loop primers LF and/or LB include, but are not limited to, primer sets A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ', R ', etc. In a specific embodiment, the kit of the invention may comprise 0.4-1.0. mu. mol/L of LF and/or LB loop primers. In a specific embodiment, the sequences of the primer sets are respectively the primers shown by FIP, BIP, F3, B3, LF and LB, or the primers with 45% or more homology to the single primer of the aforementioned sequence or its complementary strand sequence.
The kit also comprises BstDNA polymerase buffer solution, BstDNA polymerase, dNTP solution and Mg2+(MgSO4Or MgCl2) And betaine. In one embodiment, the enzymatic reaction system of the kit of the invention comprises 1 XBstDNA polymerase reaction buffer, 2-9 mmol-L Mg2+(MgSO4Or MgCl2) 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 (pH8.8), 10mmol/L KCl, 10mmol/L (NH4)2SO4，0.1％Triton X-100，2mM MgSO4. MgSO in 1 XBst DNA polymerase reaction buffer4And magnesium ion Mg in enzyme reaction system2+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 cronobacter sakazakii, or a vector comprising whole genomic DNA or partial genomic DNA of cronobacter sakazakii.
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 further comprises a color developing agent, wherein the color developing agent comprises but is not limited to calcein, SYBR Green I or hydroxynaphthol blue. When the color developing agent is calcein, the kit also comprises [ Mn2+]For example, MnCl2。
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 primer selected from the primer groups A-L, M-R, A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ' and R '. The vector contains a DNA sequence with specificity of the cronobacter sakazakii, so the vector can be applied to the research fields of microbial taxonomy, comparative genomics, evolution and the like and the application field 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 primer of primer set A, vector pBR322-B containing any one primer of primer set B, vector pBR322-R 'containing any one primer of primer set R' … …, and the like. A vector lambda phage-A containing any one of the primers of the primer set A, a vector lambda phage-B containing any one of the primers of the primer set B, … … a vector lambda phage-R 'containing any one of the primers of the primer set R', and the like.
The invention also provides application of the primer selected from any one of the primer groups A-L, M-R, A ', D ', E ', F ', G ', M ', N ', O ', P ', Q ' and R ' in detecting the cronobacter sakazakii at constant temperature.
The invention also provides application of the kit in constant-temperature detection of Cronobacter sakazakii.
The invention also provides application of the vector in constant temperature detection of Cronobacter sakazakii.
The invention provides a simple, rapid and sensitive method, primer/primer group and detection reagent/kit for detecting cronobacter sakazakii in 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 cronobacter sakazakii 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 isothermal detection method of Cronobacter sakazakii of example 7 of the present invention.
FIG. 2 shows the sensitivity of the Cronobacter sakazakii 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.