CN110423833B - Multiplex PCR method for identifying listeria monocytogenes serotype based on specific targets - Google Patents
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Abstract
The invention discloses a multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target. The method comprises the following steps: carrying out enrichment culture on the listeria monocytogenes strain to be detected to obtain an enrichment culture solution; extracting DNA of the enrichment culture solution, uniformly mixing the DNA extracting solution, the PCR primer and the PCR reactant, and performing PCR reaction to obtain a PCR reaction product; and (3) electrophoresis is carried out on the PCR reaction product, and the serotype of the bacteria to be detected is judged according to the electrophoresis result. The invention designs specific primers aiming at the newly mined specific targets lmo, 2644, LMOf2365_0118, lmo1119 and lmo0733 genes of the listeria monocytogenes serotype, and establishes a PCR identification method of the listeria monocytogenes serotype. The targets have the characteristics of high accuracy and good specificity. The method has the characteristics of high accuracy, good specificity, high identification speed, low cost and simple operation.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target.
Background
Listeria monocytogenes (Listeria monocytogenes, LM) is abbreviated as Listeria monocytogenes, and is a food-borne pathogenic bacterium for people and livestock. Listeria monocytogenes is widely found in nature, can cause contamination in many foods, and can withstand the environmental stresses common to food processing. Listeria monocytogenes is saline-alkali resistant, cold resistant, viable at 20% salt concentration, and capable of growing and reproducing at 4 ℃. Listeria monocytogenes is the only human pathogen responsible for listeriosis, primarily affecting the elderly, pregnant women, children, and immunocompromised persons. Clinical manifestations of listeriosis include meningitis, encephalitis, abortion, febrile gastroenteritis, septicemia, etc., and mortality of listeriosis is up to 30%. Therefore, most countries have strict control over listeria monocytogenes in foods, particularly in instant foods.
Listeria monocytogenes can be divided into 13 serotypes, types 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7, based on serotype responses of the thallus and flagella antigens. Of these, the 4 serotypes 1/2a, 1/2b, 1/2c, 4b are the major serotypes, and 99% of human listeriosis is caused by strains of these four serotypes. Serotype 1/2a is the most common serotype in food products, but the outbreak of listeriosis is mostly caused by strains of serotype 4b, suggesting that serotype 4b may be more virulent. The above 13 serotypes can be further divided into 5 serogroups (serogroups), types 1/2a and 3a belonging to group IIa, types 1/2b, 3b and 7 belonging to group IIb, types 1/2c and 3c belonging to group IIc, types 4b, 4d and 4e belonging to group IVb, and types 4a and 4c belonging to group L. The serotype identification of the listeria monocytogenes has important application value in monitoring epidemic strains, evaluating food safety and tracing etiology of outbreak epidemic situations.
At present, the identification method of the serotype of the listeria monocytogenes is mainly a serotype method (see the entry-exit inspection and quarantine industry standard SN-T2521-2010), and the method needs to use a pilus antigen and a thallus antigen, has higher cost, complex operation, easy cross reaction, longer identification period and more than 3 days for completing one-time identification.
In the prior patent, a PCR identification method of the serotype of the listeria monocytogenes, such as target used in the patents of CN 107746890A, CN 103602739A and the like is verified by using Primer-BLAST, and the PCR identification Primer of the serotype of the listeria monocytogenes with relatively good accuracy and specificity is found to be lacking; however, there are few reports of current literature relevant to the mining of new PCR identification targets for listeria monocytogenes serotypes. The mining of new PCR identification targets and the establishment of new PCR identification methods are helpful for improving the accuracy and the specificity of the PCR identification of the listeria monocytogenes serotype.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a multiplex PCR method for identifying listeria monocytogenes serotypes based on specific targets.
The object of the invention is achieved by at least one of the following technical solutions.
The multiplex PCR method for identifying the listeria monocytogenes serotype based on the specific target provided by the invention is a PCR identification method for identifying the listeria monocytogenes with good specificity, high identification speed, low cost and simple operation.
The invention provides a multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target, wherein a serogroup IIa, IIc, L specific target gene used in the PCR method is lmo2644, serogroup IVb and L specific target genes are LMOf2365_0118, a serogroup IIc specific target gene is lmo1119, and a listeria monocytogenes specific target gene lmo0733.
The invention discloses a group of specific targets of listeria monocytogenes serotypes, and corresponding serotype identification primers and an identification method thereof. The invention designs specific primers aiming at the newly mined specific targets lmo, 2644, LMOf2365_0118, lmo1119 and lmo0733 genes of the listeria monocytogenes serotype, and establishes a PCR identification method of the listeria monocytogenes serotype. The specific targets are obtained through comparative genomics and bioinformatics excavation, and the group of targets have the characteristics of high accuracy and good specificity. The PCR serotype identification method has the characteristics of high accuracy, good specificity, high identification speed, low cost and simplicity in operation.
The invention provides a multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target, which uses the primer sequences as follows:
lmo2644-F：5’-ACGGTATTACTTAGCGATGTGC-3’，
lmo2644-R：5’-CAAACTCAAAATAAACGCTTCAA-3’，
the corresponding characteristic DNA fragment is 669bp;
LMOf2365_0118-F：5’-ACCGACCAATGTCTACACGC-3’，
LMOf2365_0118-R：5’-TTCACGCATTCTGGCATCT-3’，
the corresponding characteristic DNA fragment is 889bp;
lmo1119-F：5’-GTGGTTCTGGTCTTGCCTTAG-3’，
lmo1119-R：5’-GATTGAGAAAGAGGGTTGAAAA-3’，
the corresponding characteristic DNA fragment is 243bp;
lmo0733-F：5’-AAAGCAATCAGAAAATCAAAAGG-3’，
lmo0733-R：5’-GTACGATTTTATACGTTCTTCCGC-3’，
the corresponding characteristic DNA fragment is 500bp.
The invention provides a multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target, which comprises the following steps of:
(1) Inoculating a listeria monocytogenes strain to be detected into a culture medium (preferably a TSB culture medium) for enrichment culture to obtain an enrichment culture solution;
(2) Extracting genome DNA of the enrichment culture solution in the step (1) by using a kit method to obtain a DNA extracting solution to be identified;
(3) Uniformly mixing the DNA extracting solution to be identified in the step (2), the PCR primer for identifying the listeria monocytogenes serotype and a PCR reactant to obtain a PCR reaction system, and then carrying out PCR reaction to obtain a PCR reaction product;
(4) Performing agarose gel electrophoresis on the PCR reaction product obtained in the step (3) to obtain an electrophoresis result diagram, and judging that the serogroup of the listeria monocytogenes strain to be detected in the step (1) is IIa and the serogroup is 1/2a or 3a if 669bp and 500bp bands appear on the electrophoresis result diagram; if only a 500bp band is present, serogroup IIb, serotype 1/2b, 3b or 7 is determined; if only 669bp, 243bp and 500bp bands are present, serogroup IIc, i.e., serotype 1/2c or 3c, is determined; if only 889bp and 500bp bands are present, serogroup IVb, i.e., serotypes 4b, 4d or 4e; if only 669bp, 889bp and 500bp bands are present, serogroup L, i.e., serotype 4a or 4c, is determined; and (3) if the bands do not appear on the electrophoresis result diagram, judging that the sample to be identified in the step (1) does not contain the listeria monocytogenes and is negative to the listeria monocytogenes.
Further, the enrichment culture in the step (1) is shaking culture in a shaking table, the temperature of the enrichment culture is 36-38 ℃, the time of the enrichment culture is 8-12 h, and the rotation speed of the shaking table of the enrichment culture is 210-230 rpm.
Preferably, the medium of step (1) is a TSB medium (i.e., trypticase Soy broth).
Preferably, the enrichment culture in the step (1) is shaking culture in a shaking table, the temperature of the enrichment culture is 37 ℃, the time of the enrichment culture is 10 hours, and the rotation speed of the shaking table of the enrichment culture is 220rpm.
Further, the DNA concentration in the DNA extract to be identified in the step (2) is 10-500 ng/. Mu.L.
Further, the PCR primers for identifying a serotype of Listeria monocytogenes in step (3) include PCR primer lmo2644, PCR primer LMOf2365_0118, PCR primer lmo0733, and PCR primer lmo1119.
Further, the PCR primers lmo2644 include a forward primer lmo2644-F and a reverse primer lmo2644-R;
the forward primer lmo2644-F is shown in SEQ ID NO:1, the reverse primer lmo2644-R is shown in SEQ ID NO:2 is shown in the figure;
the PCR primer LMOf2365_0118 comprises a forward primer LMOf2365_0118-F and a reverse primer LMOf2365_0118-R;
the forward primer LMOf2365_0118-F is shown as SEQ ID NO:3, the reverse primer LMOf2365_0118-R is shown as SEQ ID NO:4 is shown in the figure;
the PCR primer lmo1119 includes a forward primer lmo1119-F and a reverse primer lmo1119-R;
the forward primer lmo1119-F is shown in SEQ ID NO:5, the reverse primer lmo1119-R is shown in SEQ ID NO:6 is shown in the figure;
the PCR primer lmo0733 includes a forward primer lmo0733-F and a reverse primer lmo0733-R;
the forward primer lmo0733-F is shown in SEQ ID NO:7, the reverse primer lmo0733-R is shown in SEQ ID NO: shown at 8.
Further, the PCR reaction system in the step (3) comprises the following components in parts by volume:
5 parts of 10 XPCR reaction buffer;
1 part of dNTP solution;
1 part of PCR primer solution for identifying the serotype of listeria monocytogenes;
1 part of DNA extracting solution to be identified;
1 part of Taq enzyme solution;
41 parts of water;
wherein the pH value of the 10 XPCR reaction buffer is 8.2-8.4; the dNTP solution is a solution obtained by uniformly mixing dATP, dGTP, dTTP and dCTP four deoxyribonucleoside triphosphates in equal proportion with water, and the concentration of the four deoxyribonucleoside triphosphates in the dNTP solution is 10-15 mM; the PCR primer solution for identifying the serotype of the listeria monocytogenes is a solution obtained by uniformly mixing the PCR primer for identifying the serotype of the listeria monocytogenes with water, and the concentration of the primer in the PCR primer solution for identifying the serotype of the listeria monocytogenes is 8-12 mu M respectively; the Taq enzyme solution is a solution obtained by uniformly mixing Taq enzyme and water, and the concentration of the Taq enzyme solution is 2-3U/mu L; the water is sterile and nucleotide free ultrapure water (DEPC water).
Preferably, the volume of the PCR system of step (3) is 50. Mu.L.
Preferably, the PCR system of step (3) (in volume 50 μl) comprises:
10 XPCR reaction buffer 5. Mu.L, dNTP0.25mM, primer lmo2644-F, lmo2644-R, LMOf2365_0118-F, LMOf2365_0118-R, lmo1119-F, lmo1119-R, lmo0733-F, lmo0733-R each 0.2. Mu.M, template DNA 1. Mu.L, taq enzyme 2.5U, the remainder DEPC water.
Further, the conditions of the PCR reaction in the step (3) are as follows:
firstly, pre-denaturing for 2-4 min at 95-98 ℃, then performing expansion circulation, and finally extending for 4-6 min at 68-72 ℃;
the expansion cycle is: denaturation at 95-98 deg.c for 14-16 s, annealing at 52-54 deg.c for 14-16 s and extension at 68-72 deg.c for 29-31 s for 30-35 cycles.
Preferably, the conditions of the PCR reaction of step (3) are:
firstly, pre-denaturing for 3min at 95 ℃, then performing expansion circulation, and finally extending for 5min at 72 ℃;
the expansion cycle is: denaturation at 95℃for 15s, annealing at 53℃for 15s and extension at 72℃for 30s were performed for 35 cycles.
Further, the mass percentage concentration of the agarose gel in the step (4) is 1.5-3.0 wt%.
Preferably, the agarose gel electrophoresis can be performed by taking a volume of 5. Mu.L of PCR reaction product.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The multiplex PCR method for identifying the serotype of the listeria monocytogenes based on the specific target provided by the invention utilizes the PCR method to identify the serotype of the listeria monocytogenes, and has the characteristics of low cost, short period, simplicity in operation and easiness in identification result interpretation;
(2) The multiplex PCR method for identifying the serotype of the listeria monocytogenes based on the specific targets has the characteristics of high accuracy and strong specificity, and has important application value in the serotype identification of the listeria monocytogenes, especially in the monitoring of epidemic strains, food safety evaluation and etiology tracing of epidemic situations.
Drawings
FIG. 1a is a graph of partial electrophoresis results of a specific evaluation of a multiplex PCR method for identifying listeria monocytogenes serotypes based on specific targets in example 2;
FIG. 1b is a graph of another portion of the electrophoresis results of the specificity evaluation of a multiplex PCR method for identifying listeria monocytogenes serotypes based on specific targets in example 2.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or apparatus used were not manufacturer-specific and were considered conventional products commercially available.
Example 1 set up a PCR identification method of Listeria monocytogenes serotypes (the multiplex PCR method for identifying Listeria monocytogenes serotypes based on specific targets)
The complete genome sequences of the listeria monocytogenes of different serotypes are compared, a listeria monocytogenes serogroup IIa, IIc, L specific target gene lmo2644, serogroup IVb and L specific target genes LMOf2365_0118 and a serogroup IIc specific target gene lmo1119 are obtained through screening, and the listeria monocytogenes specific target gene lmo0733 is obtained through screening. Primer design was performed using the software Primer Premier 6, and the Primer sequences were determined as follows:
lmo2644-F:5'-ACGGTATTACTTAGCGATGTGC-3', as set forth in SEQ ID NO:1 is shown in the specification;
lmo2644-R:5'-CAAACTCAAAATAAACGCTTCAA-3', as set forth in SEQ ID NO:2 is shown in the figure;
lmof2365_0118-F:5'-ACCGACCAATGTCTACACGC-3', as set forth in SEQ ID NO:3 is shown in the figure;
lmof2365_0118-R:5'-TTCACGCATTCTGGCATCT-3', as set forth in SEQ ID NO:4 is shown in the figure;
lmo1119-F:5'-GTGGTTCTGGTCTTGCCTTAG-3', as set forth in SEQ ID NO:5 is shown in the figure;
lmo1119-R:5'-GATTGAGAAAGAGGGTTGAAAA-3', as set forth in SEQ ID NO:6 is shown in the figure;
lmo0733-F:5'-AAAGCAATCAGAAAATCAAAAGG-3', as set forth in SEQ ID NO: shown in figure 7;
lmo0733-R:5'-GTACGATTTTATACGTTCTTCCGC-3', as set forth in SEQ ID NO: shown at 8.
The designed primers were aligned with the NT database of NCBI using an online tool Primer-Blast to verify the accuracy of the inventive identified primers. The PCR identification primers of the present invention correctly identified the serotypes of the strain corresponding to 216 of the 217 Listeria monocytogenes genomes in the NT database. The same verification was performed using primers of the invention of Chen Jianshun et al (CN 103602739 a), and Liu Zhaoxin et al (CN 107746890 a), which were able to correctly identify serotypes of 207 and 86 genome-corresponding strains in the NR database, respectively, as shown in table 1, table 1 being an accuracy evaluation table of the serotype identification primers of the present invention. As can be seen from Table 1, the Listeria monocytogenes serotype identifying primer of the present invention has higher accuracy than the existing serotype identifying primer.
TABLE 1
PCR identification methods for the listeria monocytogenes serotypes were established using the primer pairs described above (the PCR primers described to identify the listeria monocytogenes serotypes).
The total volume of the PCR reaction system is 50 mu L, the PCR reaction system comprises 5 mu L of 10 XPCR reaction buffer solution and 1 mu L of template DNA, and the rest is DEPC water; wherein the PCR reaction system contains dNTPs 0.25mM, primers lmo2644-F, lmo2644-R, LMOf2365_0118-F, LMOf2365_0118-R, lmo1119-F, lmo1119-R, lmo0733-F and lmo0733-R each 0.2 mu M, and Taq enzyme 2.5U.
The PCR reaction procedure is as follows: pre-denaturation at 95 ℃ for 3min, after which an amplification cycle was started, each cycle being programmed to: denaturation at 95℃for 15s, annealing at 53℃for 15s, elongation at 72℃for 30s, and performing 35 cycles; finally, the extension is carried out for 5min at 72 ℃.
Judging the identification result: taking 5 mu L of PCR reaction products to carry out electrophoresis in agarose gel with the mass percent concentration of 2wt%, and judging that the serogroup of the listeria monocytogenes strain to be detected in the step (1) is IIa and the serogroup is 1/2a or 3a if only 669bp and 500bp bands appear; if only a 500bp band is present, serogroup IIb, serotype 1/2b, 3b or 7 is determined; if only 669bp, 243bp and 500bp bands are present, serogroup IIc, i.e., serotype 1/2c or 3c, is determined; if only 889bp and 500bp bands are present, serogroup IVb, i.e., serotypes 4b, 4d or 4e; if only 669bp, 889bp and 500bp bands are present, serogroup L, i.e., serotype 4a or 4c, is determined; if there is no more than one band, it is determined that Listeria monocytogenes is not present.
Example 2A multiple PCR method for identifying a Listeria monocytogenes serotype based on a specific target of the present invention
A total of 10 strains of Listeria monocytogenes, CICC 21633, CICC 21662, 242-2, CICC 21632, CICC 21634, CMCC 54002, GIM 1.347, and ATCC 19114, 2006-1 and 3877-1, respectively, of serogroup IIa, serogroup IIb, serogroup IIc, and serogroup IVb, were used for the specificity evaluation of the Listeria monocytogenes serotype PCR identification method of the present invention.
A total of 20 non-Listeria monocytogenes were used for specificity evaluation, namely, listeria incarnata (Listeria innocua), listeria (Listeria ivanovii), listeria griseum (Listeria grayi), listeria (Listeria seeligeri), listeria weissella (Listeria welshimeri), escherichia coli (Escherichia coli), pseudomonas aeruginosa (Pseudomonas aeruginosa), corynebacterium glutamicum (Corynebacterium glutamicum), proteus mirabilis (Proteus mirabilis), bacillus verdani (Bacillus wiedmannii), acinetobacter Lei Jinsi, klebsiella (Yokenella regensburgei), pseudomonas glabra (Pseudomonas granadensis), bacillus megaterium (Bacillus toyonensis), bacillus amyloliquefaciens (Paenibacillus amylolyticus), staphylococcus pini (Staphylococcus sciuri), staphylococcus succinum (Staphylococcus succinus), shewanella mandshurica (Shewanella xiamenensis), moraxella (Moraxella osloensis), streptococcus casei (Macrococcus caseolyticus), and Lactobacillus plantarum (Lactococcus garvieae).
10 strains of listeria monocytogenes and 20 strains of listeria monocytogenes are respectively inoculated into 10mL of LB liquid culture medium, the liquid culture medium is put into a shaking table for shaking culture, the rotation speed of the shaking table is 220rpm, after the bacterial growth is carried out at 37 ℃ for 12 hours, 1mL of bacterial suspension is respectively taken out, 30 parts of bacterial suspension is obtained, and 30 parts of genome DNA is obtained by extracting genome DNA by using a bacterial DNA extraction kit of Tiangen biochemical technology (Beijing) limited company.
The 30 genomic DNAs obtained by the extraction were subjected to PCR reactions in amounts of 1. Mu.L, respectively, and the PCR reaction system and the reaction procedure were as described in example 1. The PCR reaction products were subjected to electrophoresis and the results were identified as described in example 1 for the determination of the serotype of Listeria monocytogenes. The PCR identification results in example 2 are shown in Table 2, FIG. 1a and FIG. 1 b; wherein, table 2 shows the strains and the identification results thereof adopted in the specificity evaluation experiment of the PCR reaction system, M in FIG. 1a and FIG. 1b are both expressed as DL 1,000DNA markers, lanes 1-10 are Listeria monocytogenes, lanes 11-24 are non-Listeria monocytogenes, and the corresponding strains and strain names of each lane are shown in Table 2. Experimental results show that the PCR serotype identification method provided by the invention can accurately identify the serogroup to which 10 Listeria monocytogenes strains with different serotypes belong, and has higher accuracy; for 20 strains other than listeria monocytogenes, the PCR method based on multiple target identification of listeria monocytogenes serotypes provided in example 2 was all identified as negative, and therefore, the method also has higher specificity.
TABLE 2
In Table 2 "-" indicates that the identification result is non-Listeria monocytogenes.
The above examples are only preferred embodiments of the present invention, and are merely for illustrating the present invention, not for limiting the present invention, and those skilled in the art should not be able to make any changes, substitutions, modifications and the like without departing from the spirit of the present invention.
Sequence listing
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Claims (6)
1. A multiplex PCR method for identifying a serotype of listeria monocytogenes based on a specific target for use in serotyping of listeria monocytogenes in a food sample or bacterial strain of non-disease diagnostic interest, comprising the steps of:
(1) Performing enrichment culture on a food sample or a bacterial strain sample to be detected to obtain an enrichment culture solution;
(2) Extracting genome DNA of the enrichment culture solution in the step (1) by using a kit method to obtain a DNA extracting solution to be identified;
(3) Uniformly mixing the DNA extracting solution to be identified in the step (2), a PCR primer for identifying the serotype of the listeria monocytogenes and a PCR reactant to obtain a PCR reaction system, and then carrying out a PCR reaction to obtain a PCR reaction product, wherein the PCR primer for identifying the serotype of the listeria monocytogenes comprises a PCR primer lmo2644, a PCR primer LMOf2365_0118, a PCR primer lmo1119 and a PCR primer lmo0733, the PCR primer lmo2644 comprises a forward primer lmo2644-F and a reverse primer lmo2644-R, the forward primer lmo2644-F is shown as SEQ ID NO. 1, and the reverse primer lmo2644-R is shown as SEQ ID NO. 2; the PCR primer LMOf2365_0118 comprises a forward primer LMOf2365_0118-F and a reverse primer LMOf2365_0118-R, wherein the forward primer LMOf2365_0118-F is shown as SEQ ID NO. 3, and the reverse primer LMOf2365_0118-R is shown as SEQ ID NO. 4; the PCR primer lmo1119 comprises a forward primer lmo1119-F and a reverse primer lmo1119-R, wherein the forward primer lmo1119-F is shown as SEQ ID NO. 5, and the reverse primer lmo1119-R is shown as SEQ ID NO. 6; the PCR primer lmo0733 comprises a forward primer lmo0733-F and a reverse primer lmo0733-R, wherein the forward primer lmo0733-F is shown as SEQ ID NO. 7, and the reverse primer lmo0733-R is shown as SEQ ID NO. 8;
(4) Carrying out agarose gel electrophoresis on the PCR reaction product in the step (3) to obtain an electrophoresis result diagram, and judging that the serogroup of Listeria monocytogenes in the sample to be detected in the step (1) is IIa and the serogroup is 1/2a or 3a if 669bp and 500bp bands appear on the electrophoresis result diagram; if only a 500bp band is present, serogroup IIb, serotype 1/2b, 3b or 7 is determined; if only 669bp, 243bp and 500bp bands are present, serogroup IIc, i.e., serotype 1/2c or 3c, is determined; if only 889bp and 500bp bands are present, serogroup IVb, i.e., serotypes 4b, 4d or 4e; if only 669bp, 889bp and 500bp bands are present, serogroup L, i.e., serotype 4a or 4c, is determined; and (3) if the bands do not appear on the electrophoresis result diagram, judging that the sample to be detected in the step (1) does not contain the listeria monocytogenes and the listeria monocytogenes is negative.
2. The multiplex PCR method for identifying listeria monocytogenes serotype based on a specific target as defined in claim 1, wherein the culture medium used in the enrichment culture in step (1) is TSB culture medium, the enrichment culture is shaking culture in a shaking table, the temperature of the enrichment culture is 36-38 ℃, the time of the enrichment culture is 8-12 hours, and the rotation speed of the shaking table of the enrichment culture is 210-230 rpm.
3. The multiplex PCR method for identifying a serotype of listeria monocytogenes based on a specific target as in claim 1 wherein the DNA concentration in the DNA extract to be identified in step (2) is between 10 and 500ng/μl.
4. The multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target as in claim 1 wherein the PCR reaction system of step (3) comprises, in parts by volume:
wherein the pH value of the 10 XPCR reaction buffer is 8.2-8.4; the dNTP solution is a solution obtained by uniformly mixing four deoxyribonucleoside triphosphates of dATP, dGTP, dTTP and dCTP in equal proportion with water, and the concentration of the four deoxyribonucleoside triphosphates in the dNTP solution is 10-15 mM; the PCR primer solution for identifying the serotype of the listeria monocytogenes is a solution obtained by uniformly mixing the PCR primer for identifying the serotype of the listeria monocytogenes with water, and the concentration of the primer in the PCR primer solution for identifying the serotype of the listeria monocytogenes is 8-12 mu M respectively; the Taq enzyme solution is a solution obtained by uniformly mixing Taq enzyme and water, and the concentration of the Taq enzyme solution is 2-3U/mu L; the water is sterile and nucleotide free ultrapure water.
5. The multiplex PCR method for identifying a listeria monocytogenes serotype based on a specific target as in claim 1 wherein the conditions of the PCR reaction of step (3) are:
firstly, pre-denaturing for 2-4 min at 95-98 ℃, then performing expansion circulation, and finally extending for 4-6 min at 68-72 ℃;
the expansion cycle is: denaturation at 95-98 deg.c for 14-16 s, annealing at 52-54 deg.c for 14-16 s and extension at 68-72 deg.c for 29-31 s for 30-35 cycles.
6. The multiplex PCR method for identifying a serotype of listeria monocytogenes based on a specific target as claimed in claim 1 wherein the concentration of agarose gel in mass percent in step (4) is 1.5-3wt%.
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