MNP marker site, primer group and kit for identifying various swine pathogens and application of MNP marker site, primer group and kit

Abstract

The invention provides an MNP marker site for identifying various swine pathogens, a primer group, a kit and application, and belongs to the technical field of biology. And the MNP marker site comprises at least one of MNP-1 to MNP-18. The primer group comprises at least one of a first primer pair to an eighteenth primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the eighteenth primer pair and the reverse primer of the eighteenth primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 36 in a sequence table. The invention provides MNP marker sites for identifying various pig pathogens, a primer group, a kit and application, the primer group aims at 18 MNP marker sites, 1-3 marker sites are detected for each pathogen, 18 (types) pig pathogens can be identified, and the primer group has a comprehensive, efficient, sensitive and type distinguishing detection effect and is suitable for identifying various pig pathogens.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to an MNP marker site, a primer set, a kit and applications for identifying multiple porcine pathogens. Background Art

[0002] Pork is one of the world's most important meat consumption products, and pig farming is an important economic pillar industry in many regions. Pig farming is usually carried out in the form of pig herds, and pig diseases are the main factor affecting the economic benefits of pig farming. There are many pathogens that can infect pigs, and mixed infections are more common in pig diseases. Pigs are also an important medium for zoonotic pathogens. Viral pathogens are prone to mutate during the process of infecting pigs. Therefore, in the current field of farming safety and public safety, it is crucial to accurately identify and monitor the mutations of multiple pig pathogens at the same time, but this is a huge challenge for existing detection technologies.

[0003] At present, the detection technology of pig pathogens is mainly based on the nucleic acid detection technology of PCR (Polymerase Chain Reaction). However, when using PCR detection, only one marker is detected for a pig pathogen, and only one or a few pig pathogens can be monitored at a time, which makes its detection efficiency low and cannot detect the sequence information of mutations. This limits the detection of pig pathogens and is not conducive to the healthy development of the pig farming industry. Therefore, a method that can detect multiple pig pathogens at one time is urgently needed.

[0004] Public Content

[0005] In order to solve the problems of the prior art, the embodiments of the present disclosure provide a method for identifying multiple MNP marker sites, primer sets, kits and applications. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides an MNP marker site for identifying multiple pig pathogens, wherein the MNP marker site is used to identify 18 types of pig pathogens, 1-3 MNP markers are designed for each pathogen, and the MNP marker site includes at least one of MNP-1 to MNP-18. The positions of MNP-1 to MNP-18 on the reference sequence can be seen in Table 1.

[0007] On the other hand, the present disclosure provides a primer set for identifying multiple swine pathogens, which is used to detect the MNP marker site. The primer set includes: at least one pair of primer pairs from the first primer pair to the eighteenth primer pair, each of which includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the eighteenth primer pair, and the reverse primer of the eighteenth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence list.

[0008] In yet another aspect, the present disclosure provides a kit for identifying a plurality of porcine pathogens, the kit comprising the above primer set.

[0009] On the other hand, the present disclosure provides an application of the above-mentioned MNP marker site, the above-mentioned primer set or the above-mentioned kit, wherein the application includes using the MNP marker site, the above-mentioned primer set or the above-mentioned kit to identify 18 types (types) of porcine pathogens, wherein the 18 types (types) of porcine pathogens include: viruses, bacteria, fungi, special pathogens and parasites.

[0010] Specifically, the viruses include: porcine adenovirus type A, porcine adenovirus type B and porcine adenovirus type C, porcine reproductive and respiratory syndrome virus type 1, porcine reproductive and respiratory syndrome virus type 2, African swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus, rabies virus and classical swine fever virus.

[0011] Specifically, the bacteria include: Streptococcus suis and Haemophilus parasuis.

[0012] Specifically, the fungi include Cryptococcus neoformans.

[0013] Specifically, the special pathogens include: Leptospira pogepetersen and Mycoplasma hyopneumoniae.

[0014] Specifically, the parasite includes Toxoplasma gondii.

[0015] Specifically, the porcine adenovirus type A, porcine adenovirus type B and porcine adenovirus type C are three types of porcine adenovirus.

[0016] Specifically, the porcine reproductive and respiratory syndrome virus type 1 and porcine reproductive and respiratory syndrome virus type 2 are two types of porcine reproductive and respiratory syndrome virus.

[0017] Specifically, the application includes: using the MNP marker site, the primer set or the kit for identification, database construction, genetic variation monitoring and type differentiation of multiple pig pathogens.

[0018] The technical solution provided by the embodiment of the present disclosure has the following beneficial effects: the present disclosure provides an MNP marker site, primer set, kit and application for identifying multiple pig pathogens. The MNP marker site refers to a marker site that is species-specific and has multiple nucleotide polymorphisms within the species, which is screened on the genome of the target pathogen, and has high polymorphism and strong species differentiation ability. The present disclosure provides 18 MNP marker sites, which are screened from the genomes of 18 types of pig pathogens, and 1 to 3 markers are screened for each pathogen, which can specifically distinguish 18 types of pig pathogens. The primer set provided by the present disclosure has amplification compatibility, and can be amplified in a reaction system using super-multiplex PCR technology to achieve amplification of 18 MNP marker sites in a reaction system, and the second-generation sequencing platform is integrated to perform sequence analysis of the amplified products, so as to achieve identification of 18 types of pig pathogens in one reaction, with comprehensive, efficient, sensitive and accurate detection effects and type differentiation. The PCR-based nucleic acid detection technology usually uses only one pair of primers to detect one site of a pathogen in one reaction, and the fluorescence signal is detected. Compared with PCR-based nucleic acid detection technology, the present invention detects up to 3 markers for each pig pathogen, and can identify and distinguish 18 types of pathogens at a time. The detection is more efficient and accurate, and can detect sequence variations and distinguish pathogen types. It can be used for the efficient identification of various pig pathogens, genetic variation monitoring, database construction and type differentiation. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be described in further detail below.

[0020] Embodiment 1

[0021] The embodiments of the present disclosure provide an MNP marker site for identifying multiple porcine pathogens. The MNP marker site is used to identify multiple porcine pathogens. The MNP marker site includes at least one of MNP-1 to MNP-18. The positions of MNP-1 to MNP-18 on the reference sequence are shown in Table 1.

[0022] The disclosed embodiment also provides a primer set for identifying multiple swine pathogens, including: at least one pair of primer pairs from the first primer pair to the eighteenth primer pair, each primer pair including a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the eighteenth primer pair, and the reverse primer of the eighteenth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence table. The specific sequences are shown in Table 1.

[0023] Table 1 shows the primer set sequences and the locations of their corresponding MNP labeling sites

[0024]

[0025]

[0026]

[0027] Embodiment 2

[0028] The disclosed embodiments provide a kit for identifying multiple pig pathogens, the kit comprising the primer set provided in Embodiment 1.

[0029] Embodiment 3

[0030] An embodiment of the present disclosure provides an application of the above-mentioned primer set or kit, which includes using the MNP labeling site, primer set or kit to identify 18 types of pig pathogens, and the 18 types of pig pathogens include: viruses, bacteria, fungi, special pathogens and parasites.

[0031] Specifically, the viruses include: porcine adenovirus type A, porcine adenovirus type B, porcine adenovirus type C, porcine reproductive and respiratory syndrome virus type 1, porcine reproductive and respiratory syndrome virus type 2, African swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus, rabies virus and classical swine fever virus.

[0032] Specifically, the bacteria include: Streptococcus suis and Haemophilus parasuis.

[0033] Specifically, fungi include Cryptococcus neoformans.

[0034] Specifically, special pathogens include: Leptospira pogepetersen and Mycoplasma hyopneumoniae.

[0035] Specifically, parasites include Toxoplasma gondii.

[0036] Specifically, porcine adenovirus type A, porcine adenovirus type B, and porcine adenovirus type C are three types of porcine adenovirus.

[0037] Specifically, porcine reproductive and respiratory syndrome virus type 1 and porcine reproductive and respiratory syndrome virus type 2 are 2 types of porcine reproductive and respiratory syndrome virus.

[0038] In this embodiment, a primer set is used to detect 18 types (types) of positive quality control products of target pathogens (provided by Pudao (Beijing) Standard Technology Co., Ltd.). According to the number of copies of pathogens in each quality control product, the positive quality control products of the above 18 types (types) of target pathogens are mixed to prepare 3 simulated mixed samples (hereinafter referred to as simulated mixed samples). In the 3 simulated mixed samples, the number of copies of each pathogen is 1 copy / reaction, 10 copies / reaction and 100 copies / reaction, respectively, and the above 3 simulated mixed samples are named 1 copy / reaction, 10 copies / reaction and 100 copies / reaction samples, respectively. The three simulated mixed samples are detected using the kit provided in Example 2. In this embodiment, the detection method of MNP labeling is as follows: the simulated mixed sample is reverse transcribed to obtain a mixture of cDNA and DNA of the simulated mixed sample; the mixture is amplified by multiple PCR using the primer set provided in Example 1 to obtain multiple amplification products for library construction; the library is sequenced to obtain sequencing data, and an equal volume of sterile water is set as a blank control, and the blank control is named 0 copy / reaction. Three replicate libraries were constructed for each sample every day and tested continuously for 3 days, that is, 9 sets of sequencing data were obtained for each sample. The specific results are shown in Table 2.

[0039] Table 2 Evaluation results for each simulated mixed sample

[0040]

[0041] Table 2 counts the number of MNP marker sites that the detected sequences are aligned to, the number of pathogens covered, and the normalized number of sequences aligned to the MNP marker sites of pathogens in samples with different copy numbers. Among them, the normalized number of sequences refers to the number of sequences aligned to the MNP marker sites of pathogens in every 100,000 sequences. In Table 2, there is a situation where the number of detected markers is greater than the number of covered pathogens. The reason is that this embodiment detects multiple markers for some pathogens to ensure the true positive detection. When a pathogen detects multiple markers, the number of detected markers will be greater than the number of covered pathogens.

[0042] Evaluation of the stability and sensitivity of this primer set for the detection of multiple porcine pathogens

[0043] As can be seen from Table 2, when the copy number reaches 10 copies / reaction, all 18 types of pathogens are detected in 9 repetitions, indicating that the primer set has high stability in detection; when it is as low as 1 copy / reaction, not all of them can be detected. This shows that the detection sensitivity of this primer set for pig pathogens is 10 copies / reaction.

[0044] Evaluation of the reproducibility and accuracy of this primer set for multiple porcine pathogens

[0045] Based on whether the genotypes of the common detection sites in two replicates are reproducible, the reproducibility and accuracy of the MNP marker detection method for detecting porcine pathogens were evaluated. Specifically, the 9 groups of data with 100 copies / reaction in Table 2 were compared in pairs, and the results are shown in Table 3.

[0046] Table 3 shows the reproducibility and accuracy evaluation of the primer set for detecting genotypes of pig pathogens

[0047] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Reproducibility Accuracy S-1 S-2 18 18 100％ 100％ S-1 S-3 18 18 100％ 100％ S-1 S-4 18 18 100％ 100％ S-1 S-5 18 18 100％ 100％ S-1 S-6 18 18 100％ 100％ S-1 S-7 18 18 100％ 100％ S-1 S-8 18 18 100％ 100％ S-1 S-9 18 18 100％ 100％

[0048] As can be seen from Table 3, the number of MNP sites with differences in the main genotype is 0; according to the principle that the reproducible genotype between two repeated experiments is considered accurate, the accuracy rate a=1-(1-r) / 2=0.5+0.5r, r represents the reproducibility, that is, the ratio of the number of sites with reproducible main genotypes to the number of common sites. In the reproducibility test of this embodiment, the difference logarithm of the main genotype of MNP markers between different libraries and different library construction batches for each sample is 0, the reproducibility r=100%, and the accuracy rate a=100%.

[0049] Evaluation of the specificity of this primer set for porcine pathogens

[0050] As can be seen from Table 2, in the three simulated mixed samples, when the copy number of the pathogen is ≥10 copies, the sequences obtained by sequencing in the simulated samples, after sequence alignment, the MNP markers of the 18 types of pathogens provided in this example all have sequence coverage, which indicates that the MNP marker and primer set have high specificity in detecting target microorganisms in complex templates.

[0051] The threshold value of this primer set for detecting multiple swine pathogens

[0052] As shown in Table 2, most of the samples with 1 copy / reaction can detect and match the sequence of porcine pathogens. The sequence of porcine pathogens was also detected in some blank controls. Due to the extreme sensitivity of the MNP labeling detection method, data contamination during the detection process can easily lead to false positives. Therefore, in this embodiment, based on the test results of simulated samples with different copy numbers for 3 consecutive days, the following quality control plan was formulated:

[0053] 1) The amount of sequencing data is no less than 27 megabases. The calculation is based on the fact that the maximum number of MNP sites detected in each sample is 18, and the length of a sequencing fragment is 300 bases. Therefore, when the amount of data is greater than 27 megabases, most samples can ensure that the number of sequencing fragments covering each site reaches more than 5,000 times in one experiment, ensuring accurate analysis of the base sequence of each MNP site.

[0054] 2) According to the sequence number S of each porcine pathogen in the test sample and the sequence number N of the porcine pathogen in the blank control, the signal-to-noise ratio SNR=S / N of each pathogen in the sample is calculated.

[0055] Based on the sequencing data of the samples tested in Table 2, when the copy number of the pathogen is ≥10 copies, the SNR value of each pathogen is ≥10. Therefore, while ensuring accuracy and taking sensitivity into account, the judgment criterion for the primer set provided in this embodiment to be positive for pig pathogens is: when the signal-to-noise ratio of the pig pathogen in the sample is greater than 10, it is judged that the nucleic acid of the pig pathogen is detected in the sample.

[0056] Application of this primer set in the detection of multiple porcine pathogens

[0057] The primer set was used to detect the pathogens of 35 samples of pig disease materials (including diarrhea samples and swab samples) to be tested provided by Beijing Zhongke Gene Technology Co., Ltd. The sample numbers to be tested are shown in Table 4, and three negative controls with sterile water as templates were constructed at the same time. A commercial pathogen total nucleic acid extraction kit was used to prepare the pathogen total nucleic acid of each sample to be tested, and the prepared pathogen total nucleic acid of the sample to be tested was detected by qPCR. After qPCR identification, 10 positive samples of African swine fever virus were found in 35 samples to be tested. At the same time, a commercial reverse transcription kit was used to reverse transcribe the pathogen total nucleic acid of the prepared sample to be tested, and the cDNA and DNA mixed templates of the sample to be tested were obtained. The primer set provided in this embodiment was used to perform multiple PCR amplification on the cDNA and DNA mixed templates of the sample to be tested, and multiple amplification products were obtained for constructing a library, and the library was sequenced to obtain sequencing data. The results of the statistical sequencing data showed that the target pathogens were detected in all 35 samples, which were positive samples, and the detection results of the positive samples were specifically shown in Table 4.

[0058] Table 4 shows the normalized number of sequences detected by the primer set for real pig disease samples (unit: sequence)

[0059]

[0060]

[0061] Combined with Table 4, according to the above-mentioned determination threshold, 10 positive samples of African swine fever virus detected by qPCR method, using the primer set provided in Example 1, a total of 14 positive samples of African swine fever virus were detected, of which 10 were consistent with the positive samples detected by qPCR method, and 4 additional positive samples of African swine fever virus were detected, which shows that the sensitivity of the primer set in detecting African swine fever virus is not lower than that of qPCR. The pathogens detected from the 35 real pig disease samples shown in Table 4 are shown in Table 5.

[0062] Table 5 shows the statistics of pathogens detected in 35 real pig disease samples

[0063]

[0064]

[0065] Combining Table 4 and Table 5, it can be seen that in addition to African swine fever virus, in 35 samples, the primer set provided in Example 1 also detected 12 other pathogens such as porcine epidemic diarrhea virus, Haemophilus parasuis, pseudorabies virus, Mycoplasma hyopneumoniae, and Toxoplasma gondii, which shows the advantage of the primer set in comprehensive detection.

[0066] Embodiment 4

[0067] Any one pair of primers or a combination of multiple pairs of primers in the primer set is used in the detection of pig pathogens.

[0068] The primer set including 18 pairs of primer pairs provided by the present invention can realize the detection of 18 kinds (types) of target pathogens in the largest range. For applications that only focus on some of the 18 kinds (types) of pathogens, primer combinations for some pathogens can be used. For example, the 1st to 12th primer pairs in the 18 pairs of primers can realize the identification of 12 kinds (types) of viruses among the 18 kinds (types) of pig pathogens. This embodiment uses the primer set of the 1st to 12th primer pairs to re-test the 35 pig disease samples provided in Example 3, and constructs 3 negative controls with sterile water as the template. Viral pathogens were detected in 30 samples, which are viral pathogen-positive samples. The detection results of viral pathogen-positive samples are shown in Table 6.

[0069] Table 6 shows the normalized number of sequences detected by the primer set from the first primer pair to the twelfth primer pair in detecting real pig diseased material samples (unit: number)

[0070]

[0071]

[0072] Combined with Table 6, according to the above-mentioned determination threshold, the viral pathogens in all samples to be tested can be detected by the 1st to 12th primer pairs, while the test results of the samples in which the viral pathogens were not detected in Example 3 were all negative, which proves the effectiveness of any pair of primers or a combination of multiple pairs of primers in the primer set in the detection of pig pathogens.

[0073] Embodiment 5

[0074] The application includes: using the MNP marker loci, primer sets or kits for monitoring genetic variation of swine pathogen strains.

[0075] As shown in Table 4, African swine fever virus was detected in all 14 samples to be tested. The main genotypes of the MNP marker sites of African swine fever virus in each sample to be tested were compared, and it was found that the genotypes in the 14 samples to be tested were not exactly the same, and could be divided into three categories. The differences in genotypes between strains indicate that the MNP marker site, primer set or kit can be used for the detection of genetic variation of pathogens.

[0076] As a group of organisms, some individuals within the group of pig pathogens mutate, which will make the group no longer homozygous, forming a heterogeneous heterozygous group, thereby affecting the stability and consistency of the experimental microbial phenotype. This heterogeneous heterozygous group manifests as an allele type other than the main genotype of the site when the group is subjected to molecular marker detection. When the mutant individuals have not yet accumulated, they only account for a very small part of the group and manifest as a low-frequency allele type. Low-frequency allele types are often mixed with technical errors, making it difficult to distinguish with existing technologies. This embodiment detects a highly polymorphic MNP marker. Based on the probability of multiple errors occurring simultaneously is lower than the probability of one error occurring, and the technical error rate of MNP markers is significantly lower than that of SNP markers.

[0077] The genotype of the MNP marker site of African swine fever virus in sample S26, which detected more African swine fever virus sequences, was analyzed among the 35 samples to be tested as shown in Table 4. It was found that among the 62,334 sequences obtained, there were 2 different genotypes, with the number of sequences being 41,330 and 21,004, respectively, indicating that there were variations within the African swine fever virus in the same sample to be tested. It can be seen that this primer set is suitable for detecting genetic variations within the pig pathogen strain population.

[0078] Embodiment 6

[0079] Specifically, the application includes: using the MNP marker site, primer set or kit to construct a database of pig pathogens.

[0080] As shown in Table 5, after the sample to be tested is tested using the primer set, the main genotype of the MNP marker site of each pathogen in each sample to be tested is obtained, the main genotypes of the same pathogen obtained in all samples are compared, and the main genotypes with differences are entered into the database file to form the MNP fingerprint database of pig pathogens. After the main genotype of the MNP marker site of each sample or strain to be tested is compared with the constructed MNP fingerprint database, the MNP fingerprint maps of the strains with differences in the main genotype can be entered into the constructed MNP fingerprint database. Therefore, in theory, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequence of the strain to be tested, it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and updatable at any time.

[0081] Embodiment 7

[0082] Specifically, the application includes: using the MNP marker site, primer set or kit to construct fine typing of pig pathogens.

[0083] Viral pathogens among pig pathogens are classified into the following categories in taxonomy, and there are also species, subspecies and subtypes. For example, the porcine adenovirus provided in this embodiment includes adenovirus type A, adenovirus type B and adenovirus type C; porcine reproductive and respiratory syndrome virus includes porcine reproductive and respiratory syndrome virus type 1 and type 2. The primer set provided in this embodiment designs three MNP marker sites for porcine adenovirus, which are MNP markers specific to adenovirus type A, adenovirus type B and adenovirus type C, respectively. According to the detection of specific markers, pathogens can be accurately identified and finely typed. As shown in Tables 4 and 5, among the 35 samples of pig diseased materials, MNP markers specific to porcine adenovirus type A were detected in 3 samples, and MNP markers specific to porcine adenovirus type B were detected in 4 samples, thereby determining that the porcine adenoviruses in the samples were porcine adenovirus type A and type B, respectively. Similarly, the primer set provided in this embodiment screened two MNP marker sites for two types of porcine reproductive and respiratory syndrome virus, and detected one specific MNP marker for each type. Among the 35 samples shown in Table 4, 4 positive samples of porcine reproductive and respiratory syndrome virus type 2 were detected. It can be seen that the MNP marker site, primer set or kit provided in this embodiment can be used to construct the type differentiation of porcine pathogens, and thus can be used for monitoring the epidemic species (types) of porcine pathogens.

[0084] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An MNP marker site for identifying multiple porcine pathogens, characterized in that: The MNP marker site is used to identify a variety of porcine pathogens. The MNP marker site includes at least one of MNP-1 to MNP-18. The positions of MNP-1 to MNP-18 on the reference sequence are shown in the following table:

2. A primer set for identifying multiple porcine pathogens, characterized in that: The primer set includes: at least one pair of the first primer pair to the eighteenth primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the eighteenth primer pair and the reverse primer of the eighteenth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence listing.

3. A kit for identifying multiple porcine pathogens, characterized in that: The kit comprises the primer set as claimed in claim 2.

4. A use of the MNP marker site according to claim 1, the primer set according to claim 2 or the kit according to claim 3, characterized in that: The application includes using the MNP marker site, the primer set or the kit to identify multiple pig pathogens, wherein the multiple pig pathogen types include viruses, bacteria, fungi, parasites and special pathogens.

5. The use according to claim 4, characterized in that: The viruses include: porcine adenovirus type A, porcine adenovirus type B, porcine adenovirus type C, porcine reproductive and respiratory syndrome virus type 1, porcine reproductive and respiratory syndrome virus type 2, African swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus, rabies virus and classical swine fever virus.

6. The use according to claim 4, characterized in that: The bacteria include: Streptococcus suis and Haemophilus parasuis.

7. The use according to claim 4, characterized in that: The fungi include Cryptococcus neoformans.

8. The use according to claim 4, characterized in that: The special pathogens include: Leptospira pogepetersen and Mycoplasma hyopneumoniae.

9. The use according to claim 4, characterized in that: The parasites include Toxoplasma gondii.

10. The use according to claim 4, characterized in that: The application includes: using the MNP marker site, the primer set or the kit for the construction of multiple pig pathogen databases, genetic variation monitoring and type differentiation.

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