An MNP marker site, primer set, kit, and application for identifying multiple porcine pathogens.

Abstract

This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple porcine pathogens, belonging to the field of biotechnology. The MNP marker site includes at least one of MNP-1 to MNP-18. The primer set includes at least one pair of primer pairs 1 to 18, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 18, and the reverse primer of primer pair 18 are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence listing. This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple porcine pathogens. This primer set targets 18 MNP marker sites, detecting 1 to 3 marker sites for each pathogen, and can identify 18 types of porcine pathogens. It has comprehensive, efficient, sensitive, and serodifferentiating detection effects, and is suitable for identifying multiple porcine pathogens.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to an MNP marker site, primer set, kit, and application for identifying multiple porcine pathogens. Background Technology

[0002] Pork is one of the world's most consumed meats, and pig farming is a vital economic pillar in many regions. Pig farming typically operates on a herd-by-herd basis, and swine diseases are a major factor affecting the economic benefits of this industry. A variety of pathogens can infect pigs, and mixed infections are common. Pigs are also important vectors for zoonotic pathogens, and viral pathogens are prone to mutation during infection. Therefore, in the current fields of livestock safety and public safety, the simultaneous and accurate identification and mutation monitoring of multiple swine pathogens is crucial, but this presents a significant challenge to existing detection technologies.

[0003] Currently, the main technology for detecting swine pathogens is nucleic acid detection based on PCR (Polymerase Chain Reaction). However, PCR detection only detects one marker for each swine pathogen, meaning it can only monitor one or a few pathogens at a time, resulting in low detection efficiency and the inability to detect variant sequence information. This limits the detection of swine pathogens and is detrimental to the healthy development of the swine farming industry. Therefore, there is an urgent need for a method that can detect multiple swine pathogens simultaneously.

[0004] Public content

[0005] To address the problems of existing technologies, this disclosure provides an embodiment of a primer set, kit, and application for identifying various MNP marker sites. The technical solution is as follows:

[0006] On the one hand, this disclosure provides an MNP marker site for identifying multiple porcine pathogens. The MNP marker site is used to identify 18 types of porcine pathogens. One to three MNP markers are designed for each pathogen. 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 found in Table 1.

[0007] On the other hand, this disclosure provides a primer set for identifying multiple porcine pathogens and for detecting the MNP marker sites. The primer set includes at least one pair of primer pairs 1 through 18, each primer pair including a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 18th primer pair, and the reverse primer of the 18th primer pair are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence listing.

[0008] In another aspect, this disclosure provides a kit for identifying a variety of swine pathogens, the kit comprising the aforementioned primer set.

[0009] In another aspect, this disclosure provides an application of the above-mentioned MNP marker site, the above-mentioned primer set, or the above-mentioned kit, the application of which includes using the MNP marker site, the primer set, or the kit to identify 18 types of porcine pathogens, the 18 types of porcine pathogens including: 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 coronavirus type D, 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 specific pathogens include Leptospira bog Petersen and Mycoplasma hyopneumoniae.

[0014] Specifically, the parasite includes Toxoplasma gondii.

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

[0016] Specifically, 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 applications include using the MNP marker site, the primer set, or the kit for the identification, database construction, genetic variation monitoring, and type differentiation of various porcine pathogens.

[0018] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple porcine pathogens. The MNP marker site refers to a species-specific marker site screened on the genome of the target pathogen, exhibiting multiple nucleotide polymorphisms within the species, possessing high polymorphism and strong species discrimination ability. This disclosure provides 18 MNP marker sites, screened from the genomes of 18 porcine pathogens (types), with 1 to 3 markers screened for each pathogen, specifically distinguishing the 18 porcine pathogens (types). The primer set provided in this disclosure has amplification compatibility and can be amplified in a single reaction system using ultramultiplex PCR technology, achieving amplification of all 18 MNP marker sites within one reaction system. Combined with next-generation sequencing platform for sequence analysis of the amplified products, this enables the identification of 18 porcine pathogens (types) in a single reaction, providing comprehensive, efficient, sensitive, and accurate detection and type differentiation. PCR-based nucleic acid detection technology typically uses only one pair of primers to detect one site of one pathogen in a single reaction, and detects only the fluorescence signal. Compared with PCR-based nucleic acid detection technology, the present invention can detect up to 3 markers for each porcine pathogen, and identify and distinguish 18 pathogens (types) in one go. The detection is more efficient and accurate, and it can detect sequence variations and distinguish pathogen types. It can be used for efficient identification, genetic variation monitoring, database construction and type differentiation of various porcine pathogens. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below.

[0020] Example 1

[0021] This disclosure provides 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] This disclosure also provides a primer set for identifying multiple porcine pathogens, comprising at least one of primer pairs 1 to 18, each primer pair including a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 18, and the reverse primer of primer pair 18 are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence listing. Specific sequences are shown in Table 1.

[0023] Table 1 shows the primer sequence and the location of the corresponding MNP marker sites.

[0024]

[0025]

[0026]

[0027] Example 2

[0028] This disclosure provides a kit for identifying multiple swine pathogens, the kit comprising the primer set provided in Example 1.

[0029] Example 3

[0030] This disclosure provides an application of the above-mentioned primer set or kit, which includes using the MNP marker site, primer set or kit to identify 18 types of porcine pathogens, including 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 coronavirus type D, 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, the special pathogens include Leptospira bog Petersen and Mycoplasma hyopneumoniae.

[0035] Specifically, the 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 (PRRSV1) and porcine reproductive and respiratory syndrome virus type 2 (PRRSV2) are two types of porcine reproductive and respiratory syndrome virus.

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

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

[0040]

[0041] Table 2 summarizes the number of MNP marker sites aligned to detected sequences in samples with different copy numbers, the number of pathogens covered, and the normalized number of sequences aligned to MNP marker sites of pathogens. The normalized sequence number refers to the number of sequences aligned to pathogen MNP marker sites per 100,000 sequences. In Table 2, there are instances where the number of detected markers exceeds the number of pathogens covered. This is because this embodiment detects multiple markers for some pathogens to ensure true positives. When multiple markers are detected for a single pathogen, the number of detected markers will exceed the number of pathogens covered.

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

[0043] As shown in Table 2, when the copy number reaches 10 copies / reaction, all 18 pathogens (types) were detected in 9 replicates, indicating that the primer set has high detection stability; however, when the copy number is as low as 1 copy / reaction, not all pathogens can be detected. Therefore, the detection sensitivity of this primer set for swine pathogens is 10 copies / reaction.

[0044] The reproducibility and accuracy of this primer set for multiple porcine pathogens were evaluated.

[0045] The reproducibility and accuracy of the MNP marker detection method for detecting porcine pathogens were evaluated based on whether the genotypes of common detection sites could be reproduced in two replicates. Specifically, pairwise comparisons were performed on the nine groups of data with 100 copies / reaction in Table 2, and the results are shown in Table 3.

[0046] Table 3 shows the reproducibility and accuracy assessment of primer sets for detecting porcine pathogen genotypes.

[0047] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a 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 shown in Table 3, the number of MNP loci with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of reproducible major genotypes to the total number of shared loci. In the reproducibility test of this embodiment, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0049] Specificity assessment of this primer set for porcine pathogens

[0050] As shown in 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, all showed that the MNP markers of the 18 pathogens (types) provided in this embodiment were covered by the sequences. This indicates that the MNP markers and primer sets have high specificity for detecting target microorganisms in complex templates.

[0051] The primer set has a threshold for detecting multiple porcine pathogens.

[0052] Table 2 shows that sequences aligned to porcine pathogens were detected in most samples with a single copy / reaction. Porcine pathogen sequences were also detected in some blank controls. Due to the extreme sensitivity of the MNP marker 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 over three consecutive days, the following quality control scheme was formulated:

[0053] 1) The sequencing data volume should be no less than 27 megabases. The calculation is based on the fact that the maximum number of MNP sites detected per sample is 18, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 27 megabases, most samples can ensure that the number of sequencing fragments covering each site reaches more than 5000 times in one experiment, ensuring accurate analysis of the base sequence of each MNP site.

[0054] 2) Calculate the signal-to-noise ratio (SNR) of each swine pathogen in the sample based on the number of sequences S of each swine pathogen in the test sample and the number of sequences N of the swine pathogen in the blank control.

[0055] Based on the sequencing data of the samples detected 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 criterion for determining the positive result of the primer set for the pig pathogen provided in this embodiment is: when the signal-to-noise ratio of the pig pathogen in the sample is greater than 10, the nucleic acid of the pig pathogen is detected in the sample.

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

[0057] The primer set was used to detect pathogens in 35 swine samples (including diarrhea samples and swab samples) provided by Beijing Zhongke Gene Technology Co., Ltd. The sample numbers are shown in Table 4. Three negative controls using sterile water as a template were also constructed. Total pathogen nucleic acid was prepared from each sample using a commercially available pathogen total nucleic acid extraction kit. The prepared total pathogen nucleic acid was then used for African swine fever virus (ASFV) detection via qPCR. qPCR identification showed that 10 of the 35 samples were ASFV positive. Simultaneously, a commercially available reverse transcription kit was used to reverse transcribe the prepared total pathogen nucleic acid from the samples, obtaining a mixed template of cDNA and DNA. Multiplex PCR amplification was performed on the mixed template using the primer set provided in Example 1, yielding multiplex amplification products. These products were used to construct libraries, which were then sequenced to obtain sequencing data. The sequencing data showed that the target pathogen was detected in all 35 samples, classifying them as positive samples. The specific detection results for the positive samples are shown in Table 4.

[0058] Table 4 shows the normalized number of sequences detected by the primer set in real swine disease samples (unit: sequences).

[0059]

[0060]

[0061] Referring to Table 4, and according to the aforementioned judgment thresholds, of the 10 African swine fever virus (ASFV) positive samples detected by qPCR, using the primer set provided in Example 1, a total of 14 ASFV samples were detected. Of these, 10 were consistent with the positive samples detected by qPCR, and an additional 4 ASFV positive samples were detected. This indicates that the sensitivity of this primer set in detecting ASFV is no less than that of qPCR. The pathogens detected in the 35 real pig disease samples shown in Table 4 are detailed in Table 5.

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

[0063]

[0064]

[0065] As shown in Tables 4 and 5, in addition to African swine fever virus, 12 other pathogens, including porcine epidemic diarrhea virus, Haemophilus parasuis, pseudorabies virus, Mycoplasma hyopneumoniae, and Toxoplasma gondii, were also detected in the 35 samples using the primer set provided in Example 1. This demonstrates the advantage of this primer set in terms of comprehensive detection.

[0066] Example 4

[0067] Applications of any one or more primer pairs from this primer set in the detection of porcine pathogens.

[0068] The primer set provided by this invention, comprising 18 primer pairs, can achieve the detection of up to 18 target pathogens (types). For applications focusing only on a subset of the 18 pathogens (types), primer combinations targeting specific pathogens can be used. For example, primer pairs 1 through 12 of the 18 primer pairs can identify 12 viral types out of the 18 porcine pathogens (types). In this embodiment, primer pairs 1 through 12 were used to re-test 35 porcine disease samples provided in Example 3, while three negative controls were constructed using sterile water as a template. Viral pathogens were detected in 30 samples, making them positive samples for viral pathogens. The detection results for positive samples for viral pathogens are shown in Table 6.

[0069] Table 6 shows the normalized sequence counts (unit: sequences) detected in real swine disease samples using primer sets 1 through 12.

[0070]

[0071]

[0072] According to Table 6, based on the aforementioned threshold, all viral pathogens in the test samples can be detected using primer pairs 1 through 12. In Example 3, the test results of samples for which no viral pathogens were detected were all negative. This demonstrates the effectiveness of any primer pair or combination of primer pairs in this primer set in the detection of swine pathogens.

[0073] Example 5

[0074] This application includes using MNP marker sites, primer sets, or kits for monitoring genetic variations in porcine pathogen strains.

[0075] Table 4 shows that African swine fever virus was detected in all 14 samples tested. Comparison of the major genotypes of the MNP marker sites of African swine fever virus in each sample revealed that the genotypes were not entirely identical and could be divided into three main categories. The genotypic differences among the strains indicate that MNP marker sites, primer sets, or kits can be used for detecting pathogen genetic variations.

[0076] As a social organism, mutations in some individuals within a swine pathogen population can cause the population to cease homozygosity, forming a heterogeneous heterozygous population. This affects the stability and consistency of the phenotype of the experimental microorganisms. When molecular markers are detected in this heterogeneous heterozygous population, alleles other than the major genotype at the locus are present. Before the accumulation of variant individuals, they constitute only a very small portion of the population, exhibiting low-frequency alleles. Low-frequency alleles are often mixed with technical errors, making them difficult to distinguish using existing techniques. This embodiment detects highly polymorphic MNP markers. This is because the probability of multiple errors occurring simultaneously is lower than the probability of a single error occurring, and the technical error rate of MNP markers is significantly lower than that of SNP markers.

[0077] Analysis of the genotypes of African swine fever virus (ASFV) MNP marker sites in sample S26, which showed the highest number of ASFV sequences among the 35 samples tested (as shown in Table 4), revealed two distinct genotypes among the 62,334 sequences detected. These genotypes comprised 41,330 and 21,004 sequences, respectively, indicating internal variation within the ASFV population of the same sample. Therefore, this primer set is suitable for detecting genetic variation within a population of porcine pathogen strains.

[0078] Example 6

[0079] Specifically, applications include using MNP marker sites, primer sets, or kits to construct databases of porcine pathogens.

[0080] As shown in Table 5, after using this primer set to detect the test samples, the major genotypes of the MNP marker sites for each pathogen in each test sample were obtained. The major genotypes of the same pathogen obtained from all samples were compared, and the major genotypes with differences were entered into the database file, forming an MNP fingerprint database of porcine pathogens. Each time the major genotype of the MNP marker sites of a test sample or strain is compared with the constructed MNP fingerprint database, the MNP fingerprint profiles of strains with differing major genotypes can be entered into the constructed MNP fingerprint database. Therefore, theoretically, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequences of the detected strains, it is compatible with all high-throughput sequencing data and has the characteristics of being fully collaboratively built and shared, and updated at any time.

[0081] Example 7

[0082] Specifically, applications include using MNP marker sites, primer sets, or kits to construct fine typing of porcine pathogens.

[0083] Porcine pathogens, including viral pathogens, are further classified into species, subspecies, and subtypes. For example, the porcine adenoviruses provided in this embodiment include adenovirus type A, adenovirus type B, and adenovirus type C; porcine reproductive and respiratory syndrome virus (PRRSV) includes PRRSV types 1 and 2. The primer set provided in this embodiment is designed with three MNP marker sites for porcine adenoviruses, which are specific to adenovirus types A, B, and C, respectively. Based on the detection of specific markers, the pathogens can be accurately identified and finely typed. As shown in Tables 4 and 5, in 35 porcine disease samples, porcine adenovirus type A specific MNP markers were detected in 3 samples, and porcine adenovirus type B specific MNP markers were detected in 4 samples. Therefore, the porcine adenoviruses in the samples were determined to be porcine adenovirus types A and 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 (PRRSV), detecting one specific MNP marker for each type. In the 35 samples shown in Table 4, four samples were positive for PRRSV type 2. Therefore, the MNP marker sites, primer sets, or kits provided in this embodiment can be used to construct a classification system for porcine pathogens, thereby enabling the monitoring of prevalent porcine pathogen species (types).

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

Claims

1. A primer set for identifying multiple porcine pathogens, characterized in that, The primer set includes primer pairs 1 to 18, each primer pair including a forward primer and a reverse primer, the forward primer of primer pair 1, the reverse primer of primer pair 1 to the forward primer of primer pair 18 and the reverse primer of primer pair 18 are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 36 in the sequence listing.

2. A kit for identifying multiple porcine pathogens, characterized in that, The kit includes the primer set as described in claim 1.

3. The application of the primer set as described in claim 1 or the kit as described in claim 2 for identifying multiple porcine pathogens for non-disease diagnostic purposes, characterized in that, The various types of swine pathogens include viruses, bacteria, fungi, parasites, and specific pathogens. 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 coronavirus type D, rabies virus, and classical swine fever virus. The bacteria are Streptococcus suis and Haemophilus parasuis. The fungi are Cryptococcus neoformans. The specific pathogens are Leptospira borghettisen and Mycoplasma hyopneumoniae. The parasites are Toxoplasma gondii.

4. The application according to claim 3, characterized in that, The application is that the primer set or the kit is used for the construction of multiple porcine pathogen databases, genetic variation monitoring, and type differentiation.

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