Primer group and detection kit for simultaneously detecting vibrio parahaemolyticus, vibrio vulnificus and listeria monocytogenes and application thereof

Abstract

The invention relates to a primer group and a detection kit for simultaneously detecting vibrio parahaemolyticus, vibrio vulnificus and listeria monocytogenes and application of the primer group and the detection kit, and belongs to the field of microbiological detection. The detection primer group is shown as SEQ ID NO.1-18. The invention also provides a kit for simultaneously detecting the vibrio parahaemolyticus, the vibrio vulnificus and the listeria monocytogenes and application of the kit. The method disclosed by the invention can be used for simultaneously detecting whether a sample contains the vibrio parahaemolyticus and / or the vibrio vulnificus and / or the listeria monocytogenes or not; the specificity is strong, the operation is convenient and simple, and expensive instruments and equipment are not needed.

Description

Technical Field

[0001] The invention belongs to the technical field of pathogen detection, and specifically relates to a primer set and a detection kit for simultaneously detecting Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, and applications thereof. Background Art

[0002] Shellfish and other aquatic products play a vital role in the global food supply system. With the growth of the global population and the increase in demand for protein, aquaculture has become one of the important ways to ensure food safety and nutrition. In recent years, the number of cases of foodborne pathogens in aquatic products has increased dramatically due to the deterioration of the aquaculture environment and improper transportation and storage, which poses a severe challenge to public food safety. Vibrio parahaemolyticus, Vibrio vulnificus, and Listeria monocytogenes are common pathogens in aquatic products such as oysters. The presence of these pathogens not only affects the economic benefits of aquaculture, but also poses a potential risk to public health. In view of the potential threat of the above pathogens to aquaculture and human health, it is particularly important to develop efficient and accurate rapid pathogen detection technologies.

[0003] Loop-Mediated Isothermal Amplification (LAMP) is an efficient nucleic acid amplification technology that can achieve rapid and highly sensitive DNA or RNA amplification at a constant temperature. LAMP technology has been widely used in the field of molecular biology due to its simplicity, rapidity and low cost, especially in pathogen detection, genetic disease diagnosis and environmental monitoring. Multiplex LAMP technology has the advantages of short time, simple operation, high sensitivity and specificity, product visualization, etc. It can also realize the simultaneous detection of multiple pathogens, greatly shortening the time of single detection of multiple pathogens. It is a relatively promising multi-target rapid detection technology. At present, multiplex LAMP detection technology has been used in the detection of nucleic acids for a variety of mammalian diseases. Although multiplex LAMP technology has so many advantages, its research on rapid detection of foodborne pathogens in aquatic animals is still in its infancy, and multi-target LAMP detection technology that can simultaneously detect Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes has not been reported. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a loop-mediated isothermal amplification detection primer set and a detection kit and their applications that can simultaneously detect Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, have strong specificity, are easy to operate, and do not require expensive instruments and equipment.

[0005] The invention utilizes loop-mediated isothermal amplification technology, takes DNAJ gene (Genebank accession number CP022552.2) of Vibrio parahaemolyticus, tssI gene (Genebank accession number CP126699.1) of Vibrio vulnificus and HisZ gene (Genebank accession number CP021325.1) of Listeria monocytogenes as specific target genes, designs, screens and optimizes specific primers, establishes a multiple LAMP detection method, optimizes reaction conditions, adds a positive control group and a negative control group during the reaction process, and uses calcein colorimetric method and gel electrophoresis to analyze the amplified products of the detection sample group and the other two groups of controls to determine whether the sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, so as to achieve a convenient, simple, economical, rapid and sensitive detection effect, thereby achieving the purpose of the invention.

[0006] The first object of the present invention is to provide a loop-mediated isothermal amplification detection primer set for simultaneously detecting Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, wherein the detection primer set is as follows:

[0007] VP-F3: 5'-TTGCTATGGCTGCACTCG-3' (SEQ ID NO.1),

[0008] VP-B3: 5'-TCTTTTTGGCGAGCGCTTAA-3' (SEQ ID NO. 2);

[0009] VP-FIP: 5'-GGCCAGTTTGTGTTTCTGACGGCTTAAGCGGCGAAGTT

[0010] GAAGTTCCA-3' (SEQ ID NO. 3),

[0011] VP-BIP: 5'-GCGCGGTAAAGGTGTGAAAGGTTTTTAACTGGCGTTTCT

[0012] ACAACCA-3' (SEQ ID NO. 4);

[0013] VP-LF: 5'-TTTTAGGCTTACTCGTCCATCAA-3' (SEQ ID NO.5),

[0014] VP-LB: 5'-CGTGGCGGTGGTATTGGT-3' (SEQ ID NO. 6);

[0015] Vv-F3: 5'-CGAGCATTATGAATATCTGGGT-3' (SEQ ID NO.7), <h2 style=";text-align:left;direction:ltr">

[0016] <h2 style=";text-align:left;direction:ltr"> Vv-B3:5'-GGTTGAAAACAGCAGCGT-3'(SEQ ID NO.8);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0017] <h2 style=";text-align:left;direction:ltr"> Vv-FIP:5'-AGAACAAACGTATGCGTTTTTGCATCGATAGTACAAAACGAGAAATACCCA-3'(SEQID NO.9),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0018] <h2 style=";text-align:left;direction:ltr"> Vv-BIP:5'-TGTCGGCAGAAGTTTCAAATTTTGATCGATGATAACAATCGATGAGAGCAC-3'(SEQID NO.10);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0019] <h2 style=";text-align:left;direction:ltr"> Vv-LF:5'-TGTAATGATTCGATCGTG-3'(SEQ ID NO.11),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0020] <h2 style=";text-align:left;direction:ltr"> Vv-LB:5'-GCAAGCATGAAGACCCTAGC-3'(SEQ ID NO.12);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0021] <h2 style=";text-align:left;direction:ltr"> Lm-F3:5'-CGATTATGTTGTTAAAGTAGAAGGA-3'(SEQ ID NO.13),Lm-B3:5'-GCAGCATCATACAAACGATA-3'(SEQ ID NO.14);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0022] <h2 style=";text-align:left;direction:ltr"> Lm-FIP:5'-GCTTGGTAAGCTTTTTTCTGTCTTAAGCTTATATGATTT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0023] <h2 style=";text-align:left;direction:ltr"> GTTAGATGGTAATGGC-3'(SEQ ID NO.15),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0024] <h2 style=";text-align:left;direction:ltr"> Lm-BIP:5'-CTGATTATTCTTGGAGCCCTGCTTTTTACGCTACTTTCAT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0025] <h2 style=";text-align:left;direction:ltr"> CAGCA-3'(SEQ ID NO.16);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0026] <h2 style=";text-align:left;direction:ltr"> Lm-LF:5'-AATTGTCTCTGAATAATACACTTTGTCTTTTTC-3'(SEQ ID NO.17),<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0027] <h2 style=";text-align:left;direction:ltr"> Lm-LB:5'-ATATGGCCAGCGAGCGAT-3'(SEQ ID NO.18)。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0028] The second object of the present invention is to provide a multiple loop-mediated isothermal amplification detection kit for simultaneously detecting Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, comprising a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, a calcein colorimetric solution containing MnCl2, a positive control quality control product, a negative control quality control product and the detection primer set.

[0029] The positive control quality control product is preferably a mixture of plasmid DNA containing the DNAJ gene of Vibrio parahaemolyticus (Genebank accession number CP022552.2), plasmid DNA containing the tssI gene of Vibrio vulnificus (Genebank accession number CP126699.1), and plasmid DNA containing the HisZ gene of Listeria monocytogenes (Genebank accession number CP021325.1).

[0030] The negative control quality control product is ultrapure water.

[0031] The third object of the present invention is to provide a multiple loop-mediated isothermal amplification detection method for Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes for non-disease diagnosis and treatment purposes, comprising the following steps:

[0032] (1) Collecting samples and obtaining sample genomic DNA as a template;

[0033] (2) using the above-mentioned detection primer set, mixed with a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, a colorimetric solution containing MnCl2 calcein, and sample genomic DNA to form a loop-mediated isothermal amplification reaction system, and performing a loop-mediated isothermal amplification reaction;

[0034] (3) After the amplification reaction is completed, the sample is tested for the presence of Vibrio parahaemolyticus, Vibrio vulnificus, and Listeria monocytogenes by calcein fluorescence colorimetry and / or gel electrophoresis analysis.

[0035] The calcein fluorescence colorimetric method for detecting whether a sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes is specifically as follows: if the color of the amplified product is bright green, the sample contains Vibrio parahaemolyticus and / or Vibrio vulnificus and / or Listeria monocytogenes; if the color of the amplified product remains orange-yellow, the sample does not contain Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes; if the color of the amplified product remains orange-yellow, the sample does not contain Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes.

[0036] The method of detecting whether a sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes by gel electrophoresis analysis is specifically as follows: the amplified product is subjected to gel electrophoresis. If there are ladder-like bands, the sample contains Vibrio parahaemolyticus, Vibrio vulnificus or Listeria monocytogenes; if there are no ladder-like bands, the sample does not contain Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes.

[0037] The total volume of the loop-mediated isothermal amplification reaction system is 25 μL, including 2.5 μL of 10×ThermoPol Buffer, 1 μL of MgSO4 (100 mmol / L), 3.5 μL of dNTP Mix (10 mmol / L), 0.56 μL of primers Vp-FIP and Vp-BIP (50 μmol / L), 0.4 μL of primers Vp-F3 and Vp-B3 (10 μmol / L), 0.32 μL of primers Vp-LF and Vp-LB (25 μmol / L), 0.4 μL of primers Vv-FIP and Vv-BIP (50 μmol / L), and 0.56 μL of primers Vp-F3 and Vp-B3 (10 μmol / L). 3. 0.4 μL of Vv-B3 (10 μmol / L), 0.48 μL of primers Vv-LF and Vv-LB (25 μmol / L), 0.48 μL of primers Lm-FIP and Lm-BIP (50 μmol / L), 0.4 μL of primers Lm-F3 and Lm-B3 (10 μmol / L), 0.64 μL of primers Lm-LF and Lm-LB (25 μmol / L), 1 μL of 8 U Bst DNA polymerase, 0.5 μL of calcein colorimetric solution (5 mmol / L, containing 20 mmol / L MnCl2) and 1 μL of genomic DNA template, and the remainder is deionized water.

[0038] The reaction conditions of the loop-mediated isothermal amplification reaction are preferably: 58° C. for 30 min and 80° C. for 5 min.

[0039] The beneficial effects of the present invention compared with the prior art are as follows: the present invention designs a specific primer set based on the DNAJ gene of Vibrio parahaemolyticus, the tssI gene of Vibrio vulnificus and the HisZ gene of Listeria monocytogenes, which has good specificity and can simultaneously detect the presence of Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes. The present invention designs a detection kit containing the primer set and a detection method using the detection kit through loop-mediated isothermal amplification, thereby determining whether Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes are present in the detected sample. The detection kit and the detection method of the present invention have the advantages of strong specificity, convenient and simple operation, short detection time, no need for expensive instruments and equipment, and can simultaneously detect whether the sample contains three pathogens of Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, etc., and are particularly suitable for on-site detection at the grassroots level and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1This is a color analysis result diagram of the LAMP amplification product in the specificity experiment; 1: a mixture of the recombinant plasmid containing the DNAJ gene of Vibrio parahaemolyticus, the recombinant plasmid containing the tssI gene of Vibrio vulnificus and the recombinant plasmid containing the HisZ gene of Listeria monocytogenes; 2: Vibrio parahaemolyticus; 3: Vibrio vulnificus; 4: Listeria monocytogenes; 5: Vibrio alginolyticus; 6: Vibrio cholerae; 7: Vibrio alfacoum; 8: Vibrio cannelli; 9: Vibrio erucii; 10: Staphylococcus aureus; 11: ultrapure water; 1 to 4 are bright green, and 5 to 11 are orange-yellow.

[0041] Figure 2 This is the gel electrophoresis result of LAMP amplification product in specific experiment; M: standard molecular weight DL2000; 1: a mixture of recombinant plasmid containing DNAJ gene of Vibrio parahaemolyticus, recombinant plasmid containing tssI gene of Vibrio vulnificus and recombinant plasmid containing HisZ gene of Listeria monocytogenes; 2: Vibrio parahaemolyticus; 3: Vibrio vulnificus; 4: Listeria monocytogenes; 5: Vibrio alginolyticus; 6: Vibrio cholerae; 7: Vibrio alfa-ke; 8: Vibrio cannelli; 9: Vibrio erucii; 10: Staphylococcus aureus; 11: ultrapure water.

[0042] Figure 3 This is a graph of the colorimetric analysis results of the LAMP amplification products in the sensitivity experiment; 1: 1fg / ml; 2: 10fg / ml, 3: 100fg / ml, 4: 1pg / ml, 5: 10pg / ml, 6: 100pg / ml, 7: 1ng / ml, 8: 10ng / ml, 9: 100ng / ml, 10: a mixture of the recombinant plasmid containing the DNAJ gene of Vibrio parahaemolyticus, the recombinant plasmid containing the tssI gene of Vibrio vulnificus and the recombinant plasmid containing the HisZ gene of Listeria monocytogenes.

[0043] Figure 4 This is a graph of the gel electrophoresis results of the LAMP amplification products in the sensitivity experiment; M: standard molecular weight DL2000; 1: 1fg / ml; 2: 10fg / ml, 3: 100fg / ml, 4: 1pg / ml, 5: 10pg / ml, 6: 100pg / ml, 7: 1ng / ml, 8: 10ng / ml, 9: 100ng / ml, 10: a mixture of the recombinant plasmid containing the DNAJ gene of Vibrio parahaemolyticus, the recombinant plasmid containing the tssI gene of Vibrio vulnificus and the recombinant plasmid containing the HisZ gene of Listeria monocytogenes; M: standard molecular weight DL2000. DETAILED DESCRIPTION

[0044] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0045] 10×ThermoPol Buffer contains 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl2mM MgSO4, X-100, pH 8.8@25℃.

[0046] Example 1: Design and synthesis of primers According to the design principles of loop-mediated isothermal amplification primers, target gene sequences suitable for loop-mediated isothermal amplification of Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes were searched in the NCBI Genebank database, and finally the DNAJ gene (Genebank accession number CP022552.2), tssI gene (Genebank accession number CP126699.1) and HisZ gene (Genebank accession number CP021325.1) with strong specificity were selected as target genes. According to the specific regions of DNAJ gene of Vibrio parahaemolyticus, tssI gene of Vibrio vulnificus and HisZ gene of Listeria monocytogenes, according to the design principles of loop-mediated isothermal amplification primers, 5 sets of primers were designed using LAMP primer design software Primer Designer, and a set of primers with the best specificity was screened out.

[0047] The multiple loop-mediated isothermal amplification detection primers designed are:

[0048] VP-F3: 5'-TTGCTATGGCTGCACTCG-3',

[0049] VP-B3: 5'-TCTTTTTGGCGAGCGCTTAA-3';

[0050] VP-FIP: 5'-GGCCAGTTTGTGTTTCTGACGGCTTAAGCGGCGAAGTTG

[0051] AAGTTCCA-3',

[0052] VP-BIP: 5'-GCGCGGTAAAGGTGTGAAAGGTTTTTAACTGGCGTTTC

[0053] TACAACCA-3′;

[0054] VP-LF: 5'-TTTTAGGCTTACTCGTCCATCAA-3',

[0055] VP-LB: 5'-CGTGGCGGTGGTATTGGT-3';

[0056] Vv-F3: 5'-CGAGCATTATGAATATCTGGGT-3',

[0057] Vv-B3: 5'-GGTTGAAAACAGCAGCGT-3';

[0058] Vv-FIP: 5'-AGAACAAACGTATGCGTTTTGCATCGATAGTACAAAACG

[0059] AGAAATACCCA-3',

[0060] Vv-BIP: 5'-TGTCGGCAGAAGTTTCAAATTTGATCGATGATAACAATC

[0061] GATGAGAGCAC-3';

[0062] Vv-LF: 5'-TGTAATGATTCGATCGTG-3',

[0063] Vv-LB: 5'-GCAAGCATGAAGACCCTAGC-3';

[0064] Lm-F3: 5'-CGATTATGTTGTTAAAGTAGAAGGA-3',

[0065] Lm-B3: 5'-GCAGCATCATACAAACGATA-3';

[0066] Lm-FIP: 5'-GCTTGGTAAGCTTTTTTCTGTCTTAAGCTTATATGATTTG

[0067] TTAGATGGTAATGGC-3',

[0068] Lm-BIP: 5'-CTGATTATTCTTGGAGCCCTGCTTTTACGCTACT

[0069] TTCATCAGCA-3';

[0070] Lm-LF: 5'-AATTGTCTCTGAATAATACACTTTGTCTTTTTC-3',

[0071] Lm-LB: 5'-ATATGGCCAGCGAGCGAT-3'.

[0072] Example 2: Nucleic Acid Extraction

[0073] Vibrio parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes, Vibrio alginolyticus, Vibrio cholerae, Vibrio alphacrilis, Vibrio cannelli, and Vibrio erucii were inoculated into LB liquid medium and cultured overnight at 30°C; Staphylococcus aureus was inoculated into LB liquid medium and cultured overnight at 37°C. The genomes of each strain were extracted using Tiangen's bacterial genomic DNA extraction kit, and the genomic extracts were used as templates for LAMP amplification reactions.

[0074] Example 3: Construction of DNAJ, tssI and HisZ recombinant plasmids

[0075] The preparation methods of the recombinant plasmid DNAs of the DNAJ gene of Vibrio parahaemolyticus, the tssI gene of Vibrio vulnificus and the HisZ gene of Listeria monocytogenes respectively constructed are as follows: using T1-simple vector as a vector, the DNAJ gene of Vibrio parahaemolyticus, the tssI gene of Vibrio vulnificus and the HisZ gene of Listeria monocytogenes respectively as DNA target fragments, and connecting the DNAJ, tssI and HisZ target fragments to the T1-simple vector respectively through the catalysis of ligase to construct recombinant plasmids T1-DNAJ, T1-tssI and T1-HisZ, and transforming them into E.coli DH5α competent cells, screening positive clones with 100 mg / mL ampicillin, extracting the recombinant plasmids with a plasmid kit, and finally detecting by PCR and sending for sequencing to detect whether the recombinant plasmids are successfully constructed.

[0076] Example 4: The specificity of the LAMP method includes a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, a colorimetric solution containing MnCl2 calcein, a positive control quality control product, a negative control quality control product and the detection primer set.

[0077] The loop-mediated isothermal amplification reaction system of the sample was 25 μL, including: loop-mediated isothermal amplification reaction solution 7 μL (10×ThermoPol Buffer 2.5 μL, 100 mmol / LMgSO4 1 μL, 10 mmol / L dNTP Mix 3.5 μL); primers Vp-FIP and Vp-BIP (50 μmol / L) 0.56 μL each, primers Vp-F3 and Vp-B3 (10 μmol / L) 0.4 μL each, primers Vp-LF and Vp-LB (25 μmol / L) 0.32 μL each, primers Vv-FIP and Vv-BIP (50 μmol / L) 0.4 μL each, primers Vv-F3 and Vv -B3 (10μmol / L) 0.4μL each, primers Vv-LF, Vv-LB (25μmol / L) 0.48μL each, primers Lm-FIP, Lm-BIP (50μmol / L) 0.48μL each, primers Lm-F3, Lm-B3 (10μmol / L) 0.4μL each, primers Lm-LF, Lm-LB (25μmol / L) 0.64μL each; 8U Bst DNA polymerase 1μL, calcein colorimetric solution (5mmol / L calcein, containing 20mmol / L MnCl2) 0.5μL and genomic DNA template (respectively the genomic DNA obtained in Example 2) 1μL, deionized water filled to 25μL.

[0078] The loop-mediated isothermal amplification system of the positive control product was 25 μL, including: loop-mediated isothermal amplification reaction solution 7 μL (10×ThermoPol Buffer 2.5 μL, 100 mmol / LMgSO4 1 μL, 10 mmol / LdNTP Mix 3.5 μL), primers Vp-FIP and Vp-BIP (50 μmol / L) 0.56 μL each, primers Vp-F3 and Vp-B3 (10 μmol / L) 0.4 μL each, primers Vp-LF and Vp-LB (25 μmol / L) 0.32 μL each, primers Vv-FIP and Vv-BIP (50 μmol / L) 0.4 μL each, primers Vv-F3 and Vv- B3 (10 μmol / L), 0.4 μL each, primers Vv-LF and Vv-LB (25 μmol / L), 0.48 μL each, primers Lm-FIP and Lm-BIP (50 μmol / L), 0.4 μL each, primers Lm-F3 and Lm-B3 (10 μmol / L), 0.64 μL each, primers Lm-LF and Lm-LB (25 μmol / L), 1 μL of 8 U Bst DNA polymerase, 0.5 μL of calcein colorimetric solution (5 mmol / L, containing 20 mmol / L MnCl2), and 1 μL of a mixture of artificially constructed plasmids of T1-DNAJ, T1-tssI and T1-HisZ, and make up to 25 μL with deionized water.

[0079] The loop-mediated isothermal amplification reaction system of the negative control quality control product was 25 μL, including: loop-mediated isothermal amplification reaction solution 7 μL (10×ThermoPol Buffer 2.5 μL, 100mmol / L MgSO4 1 μL, 10mmol / L dNTP Mix 3.5 μL), primers Vp-FIP and Vp-BIP (50 μmol / L) 0.56 μL each, primers Vp-F3 and Vp-B3 (10 μmol / L) 0.4 μL each, primers Vp-LF and Vp-LB (25 μmol / L) 0.32 μL each, primers Vv-FIP and Vv-BIP (50 μmol / L) 0.4 μL each, primers Vv-F3 and Vv- B3 (10 μmol / L), 0.4 μL each, primers Vv-LF and Vv-LB (25 μmol / L), 0.48 μL each, primers Lm-FIP and Lm-BIP (50 μmol / L), 0.4 μL each, primers Lm-F3 and Lm-B3 (10 μmol / L), 0.64 μL each, primers Lm-LF and Lm-LB (25 μmol / L), 1 μL 8U Bst DNA polymerase, 0.5 μL of calcein colorimetric solution (5 mmol / L, containing 20 mmol / L MnCl2) and 1 μL of ultrapure water, make up to 25 μL with deionized water.

[0080] The three loop-mediated isothermal amplification systems were mixed evenly and placed in a constant temperature water bath. The reaction conditions were 58° C. for 30 min. The termination reaction was 80° C. for 5 min.

[0081] Calcein colorimetric solution (containing MnCl2) is added to the reaction system. After the reaction is completed, observe whether the color of the reaction product changes. If the color changes to bright green, it is positive; if the color changes to orange, it is negative. Figure 1 As shown in the figure, the results of calcein fluorescence colorimetric detection showed that the amplification products of Vibrio parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes and positive control quality control products were bright green, indicating positive; while the amplification reaction products of Vibrio alginolyticus, Vibrio cholerae, Vibrio alphacrilis, Vibrio cannelli, Vibrio erucii, Staphylococcus aureus and ultrapure water were orange-yellow, indicating negative.

[0082] After the loop-mediated isothermal amplification reaction was completed, 5 μL of the amplified product was spotted on a 2% agarose gel for electrophoresis detection and powered on at 180 V for 20 min. Figure 2 As shown, the electrophoresis analysis test results show that the amplification products of Vibrio parahaemolyticus, Vibrio vulnificus, Listeria monocytogenes and positive control quality control products have specific ladder-shaped bands (positive); while the amplification products of Vibrio alginolyticus, Vibrio cholerae, Vibrio alphacrilis, Vibrio cannelli, Vibrio erucicola, Staphylococcus aureus and ultrapure water have no specific bands (negative) in the electrophoresis results. This shows that the multiple loop-mediated isothermal amplification detection primers of the present invention have extremely high specificity and strong specificity, and therefore can be used to quickly detect whether a sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes.

[0083] Example 5: Sensitivity of the LAMP method

[0084] The extracted recombinant plasmids T1-DNAJ, T1-tssI and T1-HisZ were diluted to the same concentration (1 μg / ml) and mixed in equal volumes at a ratio of 1:1:1, and then diluted 10-fold with ddH2O to 1fg / ml, 10fg / ml, 100fg / ml, 1pg / ml, 10pg / ml, 100pg / ml, 1ng / ml, 10ng / ml, 100ng / ml, respectively, as DNA templates for LAMP sensitivity detection.

[0085] The loop-mediated isothermal amplification reaction system for LAMP sensitivity detection was 25 μL, including: 10×ThermoPolBuffer 2.5 μL, MgSO4 (100 mmol / L) 1 μL, dNTP Mix (10 mmol / L) 3.5 μL, primers Vp-FIP and Vp-BIP (50 μmol / L) 0.56 μL each, primers Vp-F3 and Vp-B3 (10 μmol / L) 0.4 μL each, primers Vp-LF and Vp-LB (25 μmol / L) 0.32 μL each, primers Vv-FIP and Vv-BIP (50 μmol / L) 0.4 μL each, primer Vv-F3 , Vv-B3 (10μmol / L), 0.4μL each, primers Vv-LF and Vv-LB (25μmol / L), 0.48μL each, primers Lm-FIP and Lm-BIP (50μmol / L), 0.4μL each, primers Lm-F3 and Lm-B3 (10μmol / L), 0.64μL each, primers Lm-LF and Lm-LB (25μmol / L), 1μL of 8UBst DNA polymerase, 0.5μL of calcein colorimetric solution (5mmol / L, containing 20mmol / LMnCl2), and 1μL of plasmid mixture of each diluted concentration, make up to 25μL with deionized water.

[0086] After the LAMP amplification system is mixed evenly, it is placed in a constant temperature water bath with reaction conditions of 58°C for 30 min; the termination reaction is 80°C for 5 min.

[0087] The results are as follows Figure 3 and Figure 4 As shown, the sensitivity of the loop-mediated isothermal amplification detection method of the present invention is: 100fg / ml. The results of the calcein fluorescence colorimetric method are bright green (positive) for 100fg / ml to 100ng / ml, orange-yellow (negative) for 1fg / ml and 10fg / ml; the results of 2% gel electrophoresis are specific bands for 100fg / ml to 100ng / ml (positive), and no specific bands for 1fg / ml and 10fg / ml (negative); the results of the fluorescence colorimetric method ( Figure 3 ) and gel electrophoresis results ( Figure 4 ), which shows that the sensitivity of the multiple loop-mediated isothermal amplification detection method of the present invention is 100fg / ml.

[0088] The results of the calcein fluorescence colorimetric method and the gel electrophoresis analysis were consistent, proving that the data of this method is reliable. The color of the amplified product can be used to directly determine whether the sample contains Vibrio parahaemolyticus, Vibrio vulnificus or Listeria monocytogenes, greatly shortening the detection time.

[0089] In summary, the multiple loop-mediated isothermal amplification detection primer set and detection method for Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes of the present invention can not only quickly, simply and sensitively detect Vibrio parahaemolyticus, Vibrio vulnificus or Listeria monocytogenes, but also have strong specificity and high accuracy.

Claims

1. A loop-mediated isothermal amplification detection primer set for simultaneously detecting Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, characterized in that: The detection primer set is shown in SEQ ID NO.1-18.

2. A kit for simultaneously detecting Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes, characterized in that: The kit comprises a loop-mediated isothermal amplification reaction solution, Bst DNA polymerase, a calcein colorimetric solution containing MnCl2, a positive control quality control product, a negative control quality control product and the detection primer set according to claim 1.

3. The kit according to claim 2, characterized in that: The positive control quality control product is a mixed solution of plasmid DNA containing DNAJ gene of Vibrio parahaemolyticus, plasmid DNA containing tssI gene of Vibrio vulnificus and plasmid DNA containing HisZ gene of Listeria monocytogenes; the negative control quality control product is ultrapure water.

4. Use of the detection primer set according to claim 1 or the kit according to claims 2 and 3, characterized in that: The application is to use the detection primer set or the kit to amplify through a loop-mediated isothermal amplification reaction. After the reaction is completed, calcein fluorescence colorimetry or gel electrophoresis analysis is used to detect whether the sample contains Vibrio parahaemolyticus, Vibrio vulnificus and / or Listeria monocytogenes. The purpose of the application is non-disease diagnosis and treatment.

5. The use according to claim 4, characterized in that: The calcein fluorescence colorimetric method is used to detect whether the sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes. Specifically: if the color of the amplified product is bright green, the sample contains Vibrio parahaemolyticus and / or Vibrio vulnificus and / or Listeria monocytogenes; if the color of the amplified product remains orange-yellow, the sample does not contain Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes.

6. The use according to claim 4, characterized in that: Gel electrophoresis analysis is used to detect whether the sample contains Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes. Specifically, the amplified product is subjected to gel electrophoresis. If there are ladder-like bands, the sample contains Vibrio parahaemolyticus and / or Vibrio vulnificus and / or Listeria monocytogenes; if there are no ladder-like bands, the sample does not contain Vibrio parahaemolyticus, Vibrio vulnificus and Listeria monocytogenes.

7. The use according to claim 4, characterized in that: The total volume of the loop-mediated isothermal amplification reaction system is 25 μL, including: Buffer 2.5 μL, 100 mmol / LMgSO4 1 μL, 10 mmol / L dNTP Mix 3.5μL, 50μmol / L primers Vp-FIP, Vp-BIP 0.56μL each, 10μmol / L primers Vp-F3, Vp-B3 0.4μL each, 25μmol / L primers Vp-LF, Vp-LB 0.32μL each, 50μmol / L primers Vv-FIP, Vv-BIP 0.4μL each, 10μmol / L primers Vv-F3, Vv-B3 0.4μL each, 25μmol / L primers Vv-LF, Vv-LB 0.48μL each, 50μmol / L primers Lm-FIP, Lm-BIP 0.48μL each, 10μmol / L primers Lm-F3, Lm-B3 0.4μL each, 25μmol / L primers Lm-LF, Lm-LB 0.64μL each, 8U Bst 1 μL DNA polymerase, 0.5 μL 5 mmol / L calcein colorimetric solution containing 20 mmol / L MnCl2 and 1 μL genomic DNA template, the remainder is deionized water.

8. The use according to claim 4, characterized in that: The reaction conditions of the loop-mediated isothermal amplification reaction are: 58° C. for 30 min and 80° C. for terminating the reaction for 5 min.

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