CN110890129B - Excavation method and application of Listeria monocytogenes serotype specific SNP - Google Patents
Excavation method and application of Listeria monocytogenes serotype specific SNP Download PDFInfo
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Abstract
The invention discloses an excavation method and application of Listeria monocytogenes serotype specific SNP. By the method, a plurality of serotype specific SNPs are successfully mined. Different serotype specific SNPs are combined for use, the serotype specific SNPs are arranged at the 3' tail ends of primers, so that a certain serotype is efficiently amplified, the amplification efficiency of other serotypes is extremely low, and the aim of distinguishing main serotypes is fulfilled. Therefore, the invention develops a method for identifying the main serotypes of the listeria monocytogenes from the specific base, and compared with the existing multiplex PCR method, the method realizes the reduction of the primer usage amount and has higher accuracy, and compared with the Doumith method, the consistency of the method for identifying 60 listeria monocytogenes reaches 100%.
Description
Technical Field
The invention belongs to the field of pathogenic microorganism serotype identification and detection, and relates to a method for detecting main listeria monocytogenes serotypes by using multiple PCR (polymerase chain reaction) developed on the basis of serotype specific SNP (single nucleotide polymorphism), in particular to an excavation method and application of the serotype specific SNP of listeria monocytogenes.
Background
Listeria monocytogenes is a zoonosis pathogenic bacterium, often causes serious food safety problems, causes symptoms such as meningitis, septicemia, abortion and the like after human infection, causes serious consequences to immunocompromised persons such as old people, pregnant women, children and the like, and has a fatality rate of 20-30% (refer to the literatures: radiohevich L, cossart P.Listeria monocytogenes: towards a complex picture of bits physiology and pathophysiology. Nature Reviews microbiology.2018,16 (1): 32-46). The strain has strong stress resistance, can grow in a low-temperature (4 ℃) and high-salt environment, is usually separated from chicken, fish, beef and other foods, and is a food-borne pathogenic bacterium which is mainly monitored. Along with the change of the dietary structure of China in recent years, the explosion risk of Listeria monocytogenes can be increased by large-scale eating of uncooked food and fast food, and the source tracing and monitoring of Listeria monocytogenes become more important.
Up to now, 13 serotypes have been co-detected in Listeria monocytogenes, of which types 1/2a, 1/2b, 1/2c, 4b are the most common four, accounting for over 95% of the Listeria monocytogenes isolated from clinical and food products. The traditional serotype identification method is to adopt a serum typing kit to identify according to the difference of O antigen and H antigen on the surface of thalli, and the method is time-consuming, labor-consuming and expensive. The PCR-based molecular typing method is rapid, cheap and high-throughput, can make up the deficiency of the typing of a serum kit, and at the present stage, a plurality of molecular typing methods related to the Listeria monocytogenes serotyping exist, and the serotyping of Listeria monocytogenes is realized by screening serotype specific genes and adopting the PCR method.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an excavation method of Listeria monocytogenes serotype specific SNP. By the method, a plurality of serotype specific SNPs (Single Nucleotide polymorphisms) are successfully mined. Different serotype specificity SNPs are combined for use, and an allele specificity multiplex PCR method is used for achieving the purpose of distinguishing main serotypes, and the number of primers can be saved to the maximum extent.
In addition, two specific primers are designed by simultaneously using two SNP areas of a certain gene, and a common primer is designed, so that two types can be distinguished by three primers, and the use amount of the primers is saved to the maximum extent.
Another objective of the invention is to provide a primer set for multiple PCR typing of Listeria monocytogenes serotypes.
It is another object of the present invention to provide a kit for identifying major serotypes of listeria monocytogenes.
The invention also aims to provide application of the primer group or the kit.
It is a further object of the invention to provide a method for identifying major serotypes of listeria monocytogenes.
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for excavating Listeria monocytogenes serotype specific SNP, which comprises the following steps:
(1) Firstly, 207 listeria monocytogenes genomes of known serotypes are downloaded from a biosample module of NCBI, all the downloaded genomes are in fasta format, the serotypes and the numbers are marked in each genome information, and then all the 207 genomes are written into a file in fasta format, namely genemos;
207 genes of the known serotype of the Listeria monocytogenes comprise 77 types 1/2a, 3 types 3a, 6 types 1/2c, 1 type 3c,58 types 1/2b, 1 type 3b, 1 type 7, 57 types 4b, 2 types 4a,1 type 4c;
(2) Local blast (blastn, e) is made by using all CDS and genemos of Listeria monocytogenes standard strain EGD-e<10 -200 ) And outputting in an outform-6 form, wherein the second column of the output file is a genome name with serotype labels and numbers, the fifth column is the number of unmatched bases, the results of blastn are arranged from high to low according to the sequence matching degree, and the two columns of information are combined to preliminarily screen an alternative gene containing a certain type of SNP;
(3) Local blast (blast, e) using alternative gene sequence and genemos<10 -200 ) Outputting in a default format of blastn, wherein the output result comprises sequences of the alternative genes in all genomes containing the alternative genes, completely intercepting matched strips in all genomes, corresponding to the genome names one by one, and writing all sequences into a file a in a fasta format according to the sequence of the blastn output result;
(4) And (3) opening the file a by MEGA software, displaying four bases A, T, C and G in four different colors by the MEGA software, and combining serotype labels and numbers to find out serotype specific SNP and base types.
Considering that the genome serotypes from the gene bank are not completely accurate, only SNP sites with sensitivity and specificity exceeding 85% are used as candidate targets.
It is within the scope of the claims to use this method to screen other species for other types of SNPs.
The serotypes comprise 1/2b, 3b, 7, 4b, 4d, 1/2a, 3a, 1/2c, 3c, 4a and 4c; wherein 1/2b, 3b, 7, 4b and 4d are serogroup I, 1/2a, 3a, 1/2c and 3c are serogroup II, and 4a and 4c are serogroup III.
TABLE 1 alternative targets with typing potential
Note: the underlined bases are the SNP sites used in multiplex PCR. Wherein, the 483 (A) SNP locus is at the 483 position in the Lmo1076 gene, and the A type base is a conserved and specific base type of 1/2c-3c serotype. The other same principles are adopted.
The selected multiple PCR typing target points and SNP sites are as follows:
because the whole GC content of the Listeria monocytogenes genome is low (less than 40%), a plurality of SNP sites are not suitable for designing primers, and sites suitable for designing the primers need to be selected for multiple PCR typing.
The principle of primer design: selecting genes containing two types of SNPs according to the excavated specific SNPs, selecting a region containing a plurality of continuous SNPs as far as possible, placing an SNP locus region at the 3 'end of a primer, wherein the 3' end of the primer must be a specific base, designing two specific primers, and designing a common forward or reverse primer; if the designed specific primer is a forward primer, a common reverse primer is designed, if the specific primer is a reverse primer, a common forward primer is designed, and the sequence of the common reverse primer or the common forward primer is required to be highly conserved and does not contain SNP sites; all primers require moderate GC content and cannot contain more than four G/C or A/T in succession, so that two serotypes can be separated by 3 primers, and if SNPs of multiple serotypes are contained, multiple serotypes can be separated in the method, and the quantity of the primers can be saved to the maximum.
A primer set for multiplex PCR typing of listeria monocytogenes serotypes, comprising:
(1) A primer pair for amplifying a specific gene of Listeria monocytogenes;
(2) Amplifying specific primer group of SNP locus of common gene of serogroups I and II; wherein, the common gene of serogroups I and II contains a plurality of serogroup I and II differential bases, and the types of the bases are highly conserved in the same serogroup; the specific primer group comprises a serogroup I primer F-Lm1, a serogroup II primer F-Lm2 and a common downstream reverse primer R-1-2;
(3) A specific primer group containing one 1/2c-3c type SNP and a plurality of 1/2b-3b-7 type SNPs in the amplified gene; the specific primer group comprises serotype 1/2c-3c primers R-1/2c, serotype 1/2b primers R-1/2b and a common upstream forward primer F-c-b;
preferably, the specific gene of listeria monocytogenes described in step (1) is the prfA gene; the prfA gene is referred to in the literature (Rossmanith P, krassnig M, wagner M et al, detection of Listeria monocytogenes in food use a combined expression/real-time PCR method targeting the prfA gene in research in microbiology.2006,157 (8): 763-771), the site is mature and applied to the PCR identification of Listeria monocytogenes, the gene is introduced as a control target, only the site is correctly amplified and is considered to be Listeria monocytogenes, and Listeria non-monocytogenes cannot be amplified.
Preferably, the genes shared by serogroups I and II in step (2) are Lmo1441, lmo2772, lmo2672, lmo2043, lmo0897 or Lmo0948;
preferably, the gene in step (3) is Lmo1076, lmo1078 or Lmo1188.
Further, in the above-mentioned case,
lmo1441: the gene is a gene shared by serogroups I and II, contains a plurality of serogroup I and II differential bases, and has highly conserved base types in the same serogroup, the positions 153, 155 and 156 of the gene are the 3 'ends of F-Lm2, the gene can amplify the serogroup II, the positions 630 and 633 of the gene are the 3' ends of F-Lm1, the serogroup I can be amplified, R-1-2 is F-Lm2 and F-Lm1, the common downstream reverse primer is used, and the primer sequence is highly conserved. The specific sequences of F-Lm2, F-Lm1 and R-1-2 are shown in Table 2.
Lmo1076: the kit comprises a 1/2c-3c specific base and a plurality of 1/2b-3b-7 specific bases, wherein the 483 position of the gene is the 3 'end of R-1/2c, an artificial mismatch base is introduced at the penultimate position of the 3' end to improve the specificity, the 1296, 1299 and 1302 positions are the 3 'ends of R-1/2b, an artificial mismatch is introduced at the penultimate position of the 3' end to improve the specificity, F-c-b is an upstream forward primer shared by R-1/2c and R-1/2b, and the sequence of the primer is highly conserved. The specific sequences of R-1/2c, R-1/2b and F-c-b are shown in Table 2.
TABLE 2 identification of multiple PCR primer sets for serotypes
Note: the underline marks the specific SNP site and the wavy line marks the artificial mismatch to further improve the specificity.
The invention also provides a kit for identifying the main serotypes of listeria monocytogenes, which comprises the primer group.
The primer group for multiple PCR typing of the listeria monocytogenes serotypes or the kit for identifying the main serotypes of the listeria monocytogenes are applied to identifying the serotypes of the listeria monocytogenes.
The invention also provides a method for identifying major serotypes of listeria monocytogenes comprising the steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) And (2) taking the genome DNA in the step (1) as a template, performing multiple PCR reaction by using the primer group, performing agarose gel electrophoresis on an amplification product, and analyzing the result.
Wherein F-Lm1 and R-1-2 can amplify serogroup I, F-Lm2 and R-1-2 can amplify serogroup II, R-1/2c and F-c-b can amplify 1/2c-3c type, and R-1/2b and F-c-b can amplify 1/2b-3b-7; if only the primer pair of the specific gene, the F-Lm2 and the R-1-2 in the amplification product have a target band, the amplification product is indicated to be 1/2a-3a type; if the primer pair of the specific gene in the amplification product, the F-Lm2 and R-1-2 groups, and the R-1/2c and F-c-b groups have the target bands, the target bands are indicated to be 1/2c-3c type; if only the primer pair of the specific gene, the F-Lm1 and the R-1-2 in the amplification product have a target band, indicating that the target band is 4b-4 d; if the primer pair of specific genes in the amplification product, the F-Lm1 and R-1-2 groups, and the R-1/2b and F-c-b groups have the target bands, the type is 1/2b-3 b-7.
Specifically, F-Lm1 and R-1-2 can amplify serogroup I, F-Lm2 and R-1-2 can amplify serogroup II, R-1/2c and F-c-b can amplify 1/2c-3c type, and R-1/2b and F-c-b can amplify 1/2b-3b-7; if only the prfA-F and prfA-R groups, F-Lm2 and R-1-2 groups in the amplification product have 274bp and 824bp, the amplification product is 1/2a-3a type; if the prfA-F and prfA-R groups, F-Lm2 and R-1-2 groups, and R-1/2c and F-c-b groups in the amplification product have 274bp, 824bp and 446bp, the amplification product is indicated to be 1/2c-3c type; if only the prfA-F and prfA-R groups, F-Lm1 and R-1-2 groups in the amplification product have 274bp and 346bp, the amplification product is 4b-4d type; if the amplification product has 274bp, 346bp and 1258bp of prfA-F and prfA-R groups, F-Lm1 and R-1-2 groups, R-1/2b and F-c-b groups, the amplification product is 1/2b-3b-7 type.
Preferably, the multiplex PCR reaction system described in step (2) employs a 25. Mu.L reaction system comprising 1.5U of taq enzyme (without 3' exonuclease activity), 0.3mM dNTP, 2.5. Mu.L of 10Xtaq enzyme Buffer,1.8mM MgCl 2 0.08. Mu.M of prfA-F, prfA-R, 0.2. Mu.M of F-Lm1, F-Lm2, R-1-2, 0.8. Mu.M of R-1/2c, R-1/2b, F-c-b, 2. Mu.L of DNA template ((10 ng/. Mu.L), followed by ddH 2 The amount of O was made up to 25. Mu.L.
Preferably, the conditions of the multiplex PCR reaction in step (2) are pre-denaturation at 94 ℃ for 5min, cycling reaction at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 110s,30 cycles, and finally extension at 72 ℃ for 10min, and storage at 4 ℃.
Preferably, the agarose gel electrophoresis in step (2) is 2% agarose gel electrophoresis for 35min.
60 unknown serotype wild Listeria monocytogenes isolated from commercial food were tested by first performing Multiplex PCR serotyping of 60 samples using methods reported in the literature (references: doumith M, buchrieser C, glaser P et al. Difference of the Major Listeria monocytogenes Serovars by Multiplex PCR, journal of Clinical microbiology.2004,42 (8): 3819-3822), which is more authoritative and has a 100% typing accuracy. And then the identification is carried out by the method, the typing results of the two methods are compared, and the consistency of 60 Listeria monocytogenes typing reaches 100 percent.
Compared with the prior art, the invention has the following advantages and effects:
by the mining method of the Listeria monocytogenes serotype specific SNP, multiple types of serotype specific SNPs are successfully mined, and rapid identification of main Listeria monocytogenes serotypes is realized by combining the combination of multiple SNP sites and an allele specific PCR means. The method comprises the following steps: (1) Carrying out different serotype specific SNP excavation by utilizing a strain genome information design program on NCBI; (2) designing primers based on the SNP to carry out multiplex PCR typing; (3) Typing of isolated strains in food products and comparison with international general methods. The multiple PCR typing method can maximally save the number of primers and has reliable results.
And (II) different from the conventional specific gene, the invention develops a method for identifying the main serotypes of listeria monocytogenes from a specific base, and compared with the conventional multiplex PCR method, the primer dosage is reduced. The typing accuracy is higher, and compared with the Doumith method, the typing consistency of 60 Listeria monocytogenes reaches 100%.
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FIG. 1 shows the result of allele-specific multiplex PCR electrophoresis of Listeria monocytogenes with sample numbers 1-20, wherein the number represents the sample number, which corresponds to the sample number in Table 3, and M represents marker (the size of the strip from top to bottom is 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100).
FIG. 2 shows the result of allele-specific multiplex PCR electrophoresis of food-derived Listeria monocytogenes with sample numbers 21-30, wherein the number numbers represent the sample numbers, which correspond to the sample numbers in Table 3 one by one, and M represents the marker (the size of the bands from top to bottom is 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 in order).
FIG. 3 shows the result of allele-specific multiplex PCR electrophoresis of food-derived Listeria monocytogenes with sample numbers 31-45, wherein the number represents the sample number, corresponding to the sample numbers in Table 3 one by one, and M represents the marker (the size of the bands from top to bottom is 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 in sequence).
FIG. 4 shows the result of allele-specific multiplex PCR electrophoresis for Listeria monocytogenes with sample numbers 46-60, wherein the number represents the sample number, which corresponds to the sample number in Table 3, and M represents marker (the size of the bands from top to bottom is 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1: excavation of Listeria monocytogenes serotype specific SNP
(1) Firstly, 207 genes of known serotypes of the listeria monocytogenes (including 77 types 1/2a, 3 types 3a, 6 types 1/2c, 1 type 3c,58 types 1/2b, 1 type 3b, 1 type 7, 57 types 4b and 2 types 4a,1 type 4 c) are downloaded from a biosample module of NCBI, all the downloaded genes are in fasta format, the serotypes and the numbers of the genes are marked after' >, and then all 207 genes are written into a file in fasta format, which is called genemos;
reference code:
(2) Local blast (blastn, e) is made by using all CDS (NC _ 003210.1) and genemos of Listeria monocytogenes standard strain EGD-e<10 -200 ) And outputting in an outform-6 form, wherein the second column of the output file is a genome name with serotype labels and numbers, the fifth column is the number of unmatched bases, the results of blastn can be arranged from high to low according to the sequence matching degree, and according to the output result, the serotype labels and the number of unmatched bases are combined to preliminarily screen an alternative gene containing a certain type of SNP;
reference code:
(3) Local blast (blast, e) using alternative gene sequence and genemos<10 -200 ) Outputting in a common form (in a default format of blastn), wherein the output result comprises the sequences of the candidate genes in all genomes containing the candidate genes, completely cutting off matched strips in all genomes, and matching the strips with the basesAll sequences are written into a file a together in a fasta format according to the order of output results of blastn because the group names are in one-to-one correspondence;
reference code:
(4) And (3) opening the file a by MEGA software, displaying four bases A, T, C and G in four different colors by the MEGA software, and combining serotype labels and numbers to find out serotype specific SNP sites and base types.
By the method, a great number of CDSs contain serogroup-specific SNPs, and a few CDSs contain certain serotype SNPs (see Table 1 in particular). Because the single-increment GC content is too low (the whole content is lower than 40%), a plurality of SNP sites are not suitable for designing primers, and suitable sites need to be selected. Selecting Lmo1441 and Lmo1076 as typing targets, wherein the Lmo1441 exists in serogroups I and II, contains more serogroup I and II specific SNPs, and is relatively suitable for being used as the target for distinguishing the serogroups I and II; lmo1076 contains SNPs type 1/2c-3c and 1/2b-3b-7, and is suitable for distinguishing 1/2c-3c and 1/2b-3b-7 serotypes, with specific primers shown in Table 2.
Example 2: multiplex PCR identification of Listeria monocytogenes serotypes
(1) Genome extraction
Firstly, activating and culturing the preserved Listeria monocytogenes strain by using nutrient broth, then streaking and culturing on a PALCAM (platelet-amplified polymorphism) plate to obtain a single colony, selecting the Listeria monocytogenes monoclonal to streak and amplify and culture on a blood plate, all culture conditions were 37 ℃ for 24 hours, then scraping a portion of the cells from the blood plate, extracting genomic DNA from 60 strains using a TIANGEN bacterial gene extraction kit, measuring the concentration, and diluting by an appropriate factor so that the genomic concentration was 10 ng/. Mu.L.
(2) Serotype identification
Serotype identification OF 60 samples was carried out using the currently established typing method (cf. Michel Doumith,2004.Differentiation OF the Major Listeria monocytogenes Serovars by Multiplex PCR, JOURNAL OF CLINICALMICROBIOLOGY Vol. 42, p. 8: 3819-3822). The results are shown in Table 3.
(3) Establishment of multiplex PCR System
The multiplex PCR employs a 25. Mu.L reaction system comprising 1.5U of taq enzyme (no 3' exonuclease activity), 0.3mM dNTP, 2.5. Mu.L of 10Xtaq enzyme Buffer,1.8mM MgCl 2 0.08. Mu.M prfA-F, prfA-R, 0.2. Mu.M F-Lm1, F-Lm2, R-1-2, 0.8. Mu.M R-1/2c, R-1/2b, F-c-b, 2. Mu.L of DNA template (10 ng/. Mu.L), and ddH 2 The volume of O was adjusted to 25. Mu.L. The reaction conditions are pre-denaturation at 94 ℃ for 5min, cyclic reaction at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 110s,30 cycles, extension at 72 ℃ for 10min, and preservation at 4 ℃. Finally, electrophoresis is carried out on 2% agarose for 35min. And comparing the typing identification result with the serotype identification result in the step (2). The typing and identification results are shown in FIGS. 1 to 4 and Table 3. The result shows that the typing result of the invention has higher accuracy, and compared with the Doumith method, the typing consistency of 60 Listeria monocytogenes reaches 100 percent; and realizes the reduction of the primer dosage.
TABLE 3 multiplex PCR typing results for all 60 food-borne Listeria monocytogenes samples
Note: the expression "+" indicates that the band is amplified, and the expression "-" indicates that no amplification exists, so that the serotyping method of the Listeria monocytogenes can efficiently and accurately realize the serotyping of the Listeria monocytogenes.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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<213> Artificial Sequence (Artificial Sequence)
<220>
<223> R-1-2
<400> 5
aaccagcaat tccgatatc 19
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> R-1/2c
<400> 6
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> R-1/2b
<400> 7
<210> 8
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> F-c-b
<400> 8
cctctattcc atgctaaagc ag 22
Claims (10)
1. A method for excavating Listeria monocytogenes serotype specific SNP is characterized by comprising the following steps:
(1) Firstly, 207 listeria monocytogenes genomes of known serotypes are downloaded from a biosample module of NCBI, all the downloaded genomes are in a fasta format, serotypes and numbers are marked in each genome information, and then all the 207 genomes are written into a file in the fasta format, namely genemos;
207 genes of the known serotype of the listeria monocytogenes comprise 77 types 1/2a, 3 types 3a, 6 types 1/2c, 1 type 3c,58 types 1/2b, 1 type 3b, 1 type 7, 57 types 4b, 2 types 4a,1 type 4c;
(2) Using all CDS and genemos of Listeria monocytogenes standard strain EGD-e as local blast, namely calling blast with parameter e<10 -200 And outputting in an outform-6 form, wherein the second column of the output file is a genome name with serotype labels and numbers, the fifth column is the number of unmatched bases, the results of blastn are arranged from high to low according to the sequence matching degree, and the two columns of information are combined to preliminarily screen an alternative gene containing a certain type of SNP;
(3) Using alternative gene sequence and genemos as local blast, i.e. calling blast with parameter e<10 -200 Outputting in a default format of blastn, wherein the output result comprises sequences of the alternative genes in all genomes containing the alternative genes, completely intercepting matched strips in all genomes, corresponding to the genome names one by one, and writing all sequences into a file a in a fasta format according to the sequence of the blastn output result;
(4) Opening a file a by MEGA software, wherein the MEGA software can display four bases A, T, C and G in four different colors, and serotype specific SNP and base type can be found by combining serotype labels and numbers;
wherein, only SNP loci with sensitivity and specificity exceeding 85 percent are used as alternative targets.
2. Listeria monocytogenes serotype specific SNP characterized by: the SNPs are shown in Table 1:
TABLE 1 alternative targets with typing potential
3. A primer set for multiplex PCR typing of listeria monocytogenes serotypes, comprising:
(1) A primer pair for amplifying a specific gene of Listeria monocytogenes;
(2) Amplifying specific primer group of SNP locus of common gene of serogroups I and II; wherein, the common gene of serogroups I and II contains a plurality of serogroup I and II differential bases, and the types of the bases are highly conserved in the same serogroup; the specific primer group comprises a serogroup I primer F-Lm1, a serogroup II primer F-Lm2 and a common downstream reverse primer R-1-2; wherein, the serogroup I comprises 1/2b, 3b, 7, 4b and 4d, and the serogroup II comprises 1/2a, 3a, 1/2c and 3c;
(3) A specific primer group containing one 1/2c-3c type SNP and a plurality of 1/2b-3b-7 type SNPs in the amplified gene; the specific primer group comprises serotype 1/2c-3c primers R-1/2c, serotype 1/2b primers R-1/2b and a common upstream forward primer F-c-b.
4. The primer set for Listeria monocytogenes serotype multiplex PCR typing of claim 3, wherein: the specific gene of the listeria monocytogenes strain in the step (1) is prfA gene;
the common genes of serogroups I and II in the step (2) are Lmo1441, lmo2772, lmo2672, lmo2043, lmo0897 or Lmo0948;
the gene in the step (3) is Lmo1076, lmo1078 or Lmo1188.
5. The primer set for Listeria monocytogenes serotype multiplex PCR typing according to claim 3 or 4, wherein:
(1) Primer pair of specific gene of listeria monocytogenes
prfA-F：5′-GATACAGAAACATCGGTTGGC-3′，
prfA-R：5′-GTGTAATCTTGATGCCATCAGG-3′，
(2) Listeria monocytogenes serogroup I and serogroup II specific primer group
Serogroup I primer F-Lm1:5' -ACAGAACTGTGACGCAG-3′，
Serogroup II primer F-Lm2:5' -TCACCGATTAGAAGAAGCT-3′，
Sharing a downstream reverse primer R-1-2;5 'AACCAGCAATTCCGATATCA-3',
(3) Listeria monocytogenes serotype specific primer set
Serotype 1/2c-3c primer R-1/2c:5' -GCGTTGATTTGATACCAACAT-3′，
Serotype 1/2b primer R-1/2b:5' -AAACACCCGAGTAAACGCAA-3′，
Shared upstream forward primer F-c-b:5 'CCTCTATTCCATGCTAAAGCAG-doped 3'.
6. A kit for identifying major serotypes of listeria monocytogenes, comprising: comprising the primer set according to any one of claims 3 to 5.
7. Use of the primer set for multiplex PCR typing of listeria monocytogenes serotypes according to any one of claims 3 to 5 or the kit for identifying major serotypes of listeria monocytogenes according to claim 6 for identifying listeria monocytogenes serotypes for non-disease diagnostic purposes.
8. A method for identifying major serotypes of listeria monocytogenes for non-disease diagnostic purposes, characterized by the steps of:
(1) Extracting genome DNA of a sample to be detected;
(2) Performing multiplex PCR reaction using the genomic DNA of step (1) as a template and the primer set of any one of claims 3 to 5, subjecting the amplification product to agarose gel electrophoresis, and analyzing the result.
9. The method of claim 8 for identifying major serotypes of listeria monocytogenes for non-disease diagnostic purposes, characterized in that:
F-Lm1 and R-1-2 amplify serogroup I, F-Lm2 and R-1-2 amplify serogroup II, R-1/2c and F-c-b amplify 1/2c-3c type, R-1/2b and F-c-b amplify 1/2b-3b-7; if only the primer pair of the specific gene, the F-Lm2 and the R-1-2 in the amplification product have target bands, the amplification product is indicated to be 1/2a-3a type; if the primer pair of the specific gene in the amplification product, the F-Lm2 and R-1-2 groups, and the R-1/2c and F-c-b groups have destination bands, the target bands are indicated to be 1/2c-3c type; if only the primer pair of the specific gene, the F-Lm1 and the R-1-2 in the amplification product have a target band, indicating that the target band is 4b-4 d; if the primer pair of specific genes in the amplification product, the F-Lm1 and R-1-2 groups, and the R-1/2b and F-c-b groups have destination bands, the type is 1/2b-3 b-7.
10. The method of identifying major serotypes of listeria monocytogenes for non-disease diagnostic purposes according to claim 8 or 9, characterized in that:
F-Lm1 and R-1-2 amplify serogroup I, F-Lm2 and R-1-2 amplify serogroup II, R-1/2c and F-c-b amplify 1/2c-3c type, R-1/2b and F-c-b amplify 1/2b-3b-7; if only the prfA-F and prfA-R groups, F-Lm2 and R-1-2 groups in the amplification product have 274bp and 824bp, the amplification product is 1/2a-3a type; if the prfA-F and prfA-R groups, F-Lm2 and R-1-2 groups, and R-1/2c and F-c-b groups in the amplification product have 274bp, 824bp and 446bp, the amplification product is indicated to be 1/2c-3c type; if only the prfA-F and prfA-R groups, F-Lm1 and R-1-2 groups in the amplification product have 274bp and 346bp, the amplification product is 4b-4d type; if the prfA-F and prfA-R groups, F-Lm1 and R-1-2 groups, R-1/2b and F-c-b groups in the amplification product have 274bp, 346bp and 1258bp, the amplification product is 1/2b-3b-7 type.
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