Porcine pseudorabies virus genotyping probe primer set, typing kit and typing method

Abstract

The present invention provides a porcine pseudorabies virus genotyping probe primer set, a genotyping kit and a genotyping method, belonging to the technical field of gene detection. The present invention provides a set of porcine pseudorabies virus genotyping probe primer set, including a pair of universal primers and genotyping TaqMan probes. The genotyping probe primer set of the present invention has a good linear relationship within the dilution range (10<supgt;1< / supgt> copies / μL to 10<supgt;6< / supgt> copies / μL), and the lowest detection limit is 10<supgt;1< / supgt> copies / μL, having good sensitivity; there is no cross-reaction with other viruses, and the specificity is good; the coefficient of variation of each dilution within the group and between groups is less than 3%, and the repeatability is good. Therefore, the genotyping probe primer set of the present invention can stably identify the PRV genotype.

Description

Technical Field

[0001] The invention belongs to the technical field of gene detection, and in particular relates to a porcine pseudorabies virus gene typing probe primer set, a typing kit and a typing method. Background Art

[0002] Pseudorabies virus (PRV), also known as porcine herpesvirus type I, belongs to the α-herpesvirus subfamily of the herpesviridae family. The virus particles are oval or round, the genome is about 143,000 base pairs, and the GC content is as high as 73%. The virus uses pigs as its natural host and can infect other livestock or wild animals, causing acute infectious diseases characterized by central nervous system diseases. Because its symptoms are similar to rabies, it is named pseudorabies. The disease causes great harm to the pig farming industry. The younger the pig is, the more susceptible it is to pseudorabies virus. The mortality rate of piglets in the delivery room is nearly 100%, and infection of adult pigs will cause reproductive disorders, such as causing sows to abort, produce stillbirths or mummies, and cause boars to have abnormal sexual function and reduced reproductive capacity, which will lead to a decline in many indicators such as growth rate, breeding rate and reproduction rate of pig farms. PRV has caused huge economic losses to the pig farming industry worldwide, and WOAH has classified it as a Class B infectious disease.

[0003] The large-scale use of Bartha-K61, a pseudorabies attenuated vaccine, has effectively curbed the outbreak and spread of pseudorabies. However, since the second half of 2011, many large-scale pig farms in China that have been immunized with Bartha-K61 have experienced outbreaks of pseudorabies in pigs. Studies have found that the virulence of the newly popular PRV in China is significantly stronger than that of the classic strain, and it is named genotype II. Traditional inactivated and attenuated vaccines have great limitations in their cross-protection against different genotypes of PRV. Therefore, it is particularly important to carry out rapid and highly sensitive identification and diagnosis of pseudorabies virus in pigs. Summary of the invention

[0004] The invention provides a porcine pseudorabies virus gene typing probe primer set, a typing kit and a typing method, which can accurately, specifically and highly sensitively distinguish between type I and type II PRV.

[0005] The present invention provides a set of porcine pseudorabies virus genotyping probe primer sets, comprising a pair of universal primers and a genotyping TaqMan probe, wherein the nucleotide sequences of the universal primers are shown in SEQ ID No. 1 to SEQ ID No. 2;

[0006] The genotyping TaqMan probe includes at least one of the following: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.5.

[0007] Preferably, each of the genotyping TaqMan probes is labeled with a different fluorescent group at the 5' end and a different quenching group at the 3' end.

[0008] Preferably, the fluorescent group is selected from Texas Red, 6-FAM or CY5; and the quenching group is selected from BHQ2, MGB or BHQ3.

[0009] The present invention also provides a detection kit for swine pseudorabies virus genotype I, comprising a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe comprises one probe or two probes, when it is one probe, the genotyping TaqMan probe is a type I probe; when it is two probes, the genotyping TaqMan probe is a type I probe and a universal probe;

[0010] The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2;

[0011] The nucleotide sequence of the type I probe is shown in SEQ ID No. 3;

[0012] The nucleotide sequence of the universal probe is shown in SEQ ID No.5.

[0013] The present invention also provides a detection kit for swine pseudorabies virus genotype II, comprising a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe comprises one probe or two probes, when it is one probe, the genotyping TaqMan probe is a type II probe; when it is two probes, the genotyping TaqMan probe is a type II probe and a universal probe;

[0014] The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2;

[0015] The nucleotide sequence of the type II probe is shown in SEQ ID No.4;

[0016] The nucleotide sequence of the universal probe is shown in SEQ ID No.5.

[0017] The present invention also provides a kit for genotyping of porcine pseudorabies virus, comprising a pair of universal primers and three genotyping TaqMan probes, wherein the nucleotide sequences of the universal primers are shown in SEQ ID No. 1 to SEQ ID No. 2;

[0018] The genotyping TaqMan probes include: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.5.

[0019] The present invention also provides a method for genotyping porcine pseudorabies virus with a single probe, comprising using a target genomic nucleic acid as a template, mixing it with the universal primers and genotyping TaqMan probes in the above-mentioned detection kit to configure a fluorescent quantitative PCR typing detection system, performing a PCR reaction, and then genotyping the porcine pseudorabies virus according to the amplification result.

[0020] Preferably, the fluorescent quantitative PCR typing detection system, in 20 μL, includes: 10 μL of 2×AceQ qPCR ProbeMasterMix, 2 μL of template, 0.9 μL of each universal primer, 0.4 μL of each genotyping TaqMan probe, and the balance of ddH2O.

[0021] Preferably, the PCR reaction procedure includes: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles.

[0022] The present invention also provides a method for genotyping porcine pseudorabies virus using multiple probes, comprising using a target genomic nucleic acid as a template, mixing it with the universal primers and genotyping TaqMan probes in the above-mentioned detection kit to configure a fluorescent quantitative PCR typing detection system, performing a PCR reaction, and then genotyping the porcine pseudorabies virus according to the amplification results.

[0023] Beneficial effect: The present invention provides a set of porcine pseudorabies virus genotyping probe primer sets, including a pair of universal primers and a genotyping TaqMan probe, wherein the genotyping probe primer set is designed based on the gN gene, wherein the PRV genotype I standard curve is Y=-3.492X+39.921 (Y: Ct value, X: logarithmic value of plasmid copy number), and the correlation coefficient R 2 =0.9998. The standard curve of PRV genotype II is Y=-3.5378X+44.095, and the correlation coefficient is R 2 =0.9998. That is, the genotyping probe primer set of the present invention has a dilution of (10 1 copies / μL~10 6 The linear relationship was good in the range of 10 copies / μL, and the minimum detection limit was 10 1 The sensitivity is 300 copies / μL, and there is good cross reaction with other viruses, and the specificity is good; the coefficient of variation of each dilution within and between groups is less than 3%, and the repeatability is good. Therefore, the genotyping probe primer set of the present invention can stably identify the PRV genotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a phylogenetic tree constructed using IQTREE software;

[0025] Figure 2 The schematic diagram is for designing the sequence of primers and probes based on the gN gene;

[0026] Figure 3 The standard curves for PRV type I and II;

[0027] Figure 4 This is a comparison of the sensitivity of single-probe and three-probe typing. The green line in the figure is a single probe, and the red line is a three-probe;

[0028] Figure 5 This is a diagram showing the specificity detection results of the genotyping probe primer set of the present invention. DETAILED DESCRIPTION

[0029] The present invention provides a set of porcine pseudorabies virus genotyping probe primer sets, comprising a pair of universal primers and a genotyping TaqMan probe, wherein the nucleotide sequences of the universal primers are shown in SEQ ID No. 1 to SEQ ID No. 2;

[0030] The genotyping TaqMan probe includes at least one of the following: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.5.

[0031] The genotyping probe primer set of the present invention is preferably designed based on the gN gene of PRV, and positions 105-123, 400-418 and 195-211 of the gN gene are conserved in the two genotypes of PRV, and the first two are selected as upstream and downstream primers, respectively, and the latter is used as a universal probe; among them, positions 321-340 of the gN gene are highly conserved and sequence-specific in PRV genotype I and genotype II, respectively, so they are selected as PRV genotype I probes, and similarly, positions 325-340 are selected as PRV genotype II probes.

[0032] The sequence of the genotyping probe primer set of the present invention is preferably as follows:

[0033] Upstream primer (SEQ ID No. 1): gN-F: GCGACGAGCATCGCGTATC;

[0034] Downstream primer (SEQ ID No. 2): gN-R: GGCTGATCCACCGGGACTC;

[0035] Type I probe (SEQ ID No. 3): Texas Red-AGCGGCCCCTCGGTGGAGAC-BHQ2;

[0036] Type II probe (SEQ ID No. 4): 6-FAM-AGCGACCCCTCGGAAA-MGB;

[0037] Universal probe (SEQ ID No. 5): CY5-AGCAGCCGCCCTCCGTC-BHQ3.

[0038] The present invention preferably labels each of the genotyping TaqMan probes with a different fluorescent group at the 5' end and a different quenching group at the 3' end. The fluorescent group of the present invention is selected from Texas Red, 6-FAM or CY5; the quenching group is selected from BHQ2, MGB or BHQ3. The universal primer of the present invention can be used to amplify a fragment of the gN gene, and the universal probe can target and bind to the gN gene, thereby determining whether the target contains the PRV gene.

[0039] The present invention also provides a detection kit for swine pseudorabies virus genotype I, comprising a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe comprises one probe or two probes, when it is one probe, the genotyping TaqMan probe is a type I probe; when it is two probes, the genotyping TaqMan probe is a type I probe and a universal probe;

[0040] The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2;

[0041] The nucleotide sequence of the type I probe is shown in SEQ ID No. 3;

[0042] The nucleotide sequence of the universal probe is shown in SEQ ID No.5.

[0043] When performing genotyping, the present invention may add a universal probe to the detection system, or may not add a universal probe. The fluorescent quantitative PCR typing detection system, in 20 μL, includes: 10 μL of 2×AceQ qPCR Probe MasterMix, 2 μL of template, 0.9 μL of universal primers, 0.4 μL of genotyping TaqMan probes, and the remainder of ddH2O. The PCR reaction procedure of the present invention preferably includes: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles. After the PCR reaction, the present invention determines whether PRV belongs to type I according to fluorescence.

[0044] The present invention also provides a detection kit for swine pseudorabies virus genotype II, comprising a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe comprises one probe or two probes, when it is one probe, the genotyping TaqMan probe is a type II probe; when it is two probes, the genotyping TaqMan probe is a type II probe and a universal probe;

[0045] The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2;

[0046] The nucleotide sequence of the type II probe is shown in SEQ ID No.4;

[0047] The nucleotide sequence of the universal probe is shown in SEQ ID No.5.

[0048] The contents and detection method of the detection kit of the present invention are preferably the same as those described above and will not be described in detail herein.

[0049] The present invention also provides a kit for genotyping of porcine pseudorabies virus, comprising a pair of universal primers and three genotyping TaqMan probes, wherein the nucleotide sequences of the universal primers are shown in SEQ ID No. 1 to SEQ ID No. 2;

[0050] The genotyping TaqMan probes include: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.5.

[0051] The present invention also provides a method for genotyping porcine pseudorabies virus with a single probe, comprising using a target genomic nucleic acid as a template, mixing it with the universal primers and genotyping TaqMan probes in the above-mentioned detection kit to configure a fluorescent quantitative PCR typing detection system, performing a PCR reaction, and then genotyping the porcine pseudorabies virus according to the amplification result.

[0052] When the present invention uses a single probe for PRV typing detection, the fluorescent quantitative PCR typing detection system preferably includes 20 μL: 10 μL of 2×AceQ qPCRProbe MasterMix, 2 μL of template, 0.9 μL of universal primers, 0.4 μL of genotyping TaqMan probe, and the balance of ddH2O. The PCR reaction program of the present invention preferably includes: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles.

[0053] The present invention also provides a method for genotyping porcine pseudorabies virus using multiple probes, comprising using a target genomic nucleic acid as a template, mixing it with the universal primers and genotyping TaqMan probes in the above-mentioned detection kit to configure a fluorescent quantitative PCR typing detection system, performing a PCR reaction, and then genotyping the porcine pseudorabies virus according to the amplification results.

[0054] When the present invention uses triple probes for PRV typing detection, the fluorescent quantitative PCR typing detection system preferably includes 20 μL: 10 μL of 2×AceQ qPCRProbe MasterMix, 2 μL of template, 0.9 μL of universal primers, 0.4 μL of genotyping TaqMan probes, and the balance of ddH2O. The PCR reaction program of the present invention preferably includes: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles.

[0055] To further illustrate the present invention, the porcine pseudorabies virus genotyping probe primer set, typing kit and typing method provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0056] The sources of the reagents and instruments used in the examples of the present invention are as follows:

[0057] AceQ qPCR Probe MasterMix (P212-03) was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; TSINGKE TSV-007VS pClone007 VersatileSimple Vector Kit (TSV-007VS) was purchased from Beijing Qingke Biotechnology Co., Ltd.; TIANamp Virus DNA / RNA Fast Kit (DP315-F) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Bio-Rad CFX 96 fluorescence quantitative PCR instrument was purchased from Bio-Rad Corporation in the United States.

[0058] The viral genome and plasmid used in the examples of the present invention are as follows:

[0059] The viral genomes of PRV-DX, PRV-BarthaK61, porcine reproductive and respiratory syndrome virus (PRRSV), porcine deltacoronavirus (PDCoV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV) and porcine circovirus type 2 (PCV2); the plasmid containing the full-length genome of porcine circovirus type 3 (PCV3) was constructed by synthesizing MH018246 into the HindIII site of the pUC57 plasmid through homologous recombination, and the plasmid containing the full-length genome of porcine circovirus type 4 (PCV4) was constructed by synthesizing NC_055580 into the HindIII site of the pUC57 plasmid through homologous recombination.

[0060] Example 1

[0061] All published PRV genome sequences were obtained from the NCBI database, and sequence alignment was completed using MAFFT software. Phylogenetic evolutionary trees were constructed using IQTREE software to categorize PRV sequences. The evolutionary tree results showed that PRV can be divided into genotype I and genotype II, of which genotype I and genotype II PRV have 17 and 48 sequences, respectively ( Figure 1 ). According to the stable differential gene sequences of genotype I and genotype II PRV ( Figure 2 ), a pair of universal primers and three probes (SEQ ID No.1 to SEQ ID No.5) were designed, which were PRV gene type I probe, PRV gene type II probe and PRV universal probe. The primers were synthesized by Zhejiang Shangya Biotechnology Co., Ltd., and the probes were synthesized by Anhui General Biotechnology Co., Ltd.

[0062] exist Figure 2 It was found that positions 105-123, 400-418 and 195-211 of the gN gene were conserved in both genotypes of PRV, and the first two were selected as upstream and downstream primers, respectively, and the latter was used as a universal probe; among them, positions 321-340 of the gN gene were highly conserved and sequence-specific in PRV genotype I and genotype II, respectively, so they were selected as PRV genotype I probes. Similarly, positions 325-340 were selected as PRV genotype II probes.

[0063] The partial sequence of gN gene related to PRV typing (from PRV-DX and PRV-Bartha K61 preserved in the laboratory, disclosed in the literature A wide extent of inter-strain diversity in virulent and vaccinestrains of alphaherpesviruses. Szpara ML, et al. PLoS Pathog, 2011 Oct. PMID22022263) was blunt-ended with pClone007 and ligated to the vector pUC19 to construct recombinant plasmids pUC-DX5 and pUC-BT5 as templates for subsequent experiments.

[0064] Example 2

[0065] Establishment and optimization of TaqMan multi-probe fluorescence quantitative PCR typing detection system for PRV

[0066] The primers and probes designed in Example 1 were used to establish the detection system. The PCR detection system was: 10 μL of 2×AceQqPCRProbe MasterMix, 2 μL of standard plasmid, 0.9 μL of 10 μM upstream and downstream primers, 0.4 μL of 10 μM probes, and finally 5 μL of ddH2O was used to make up to 20 μL. The PCR amplification conditions were: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles.

[0067] Example 3

[0068] Specificity test

[0069] PDCoV, CSFV, PRRSV, PEDV, PSFV, PCV2 viral DNA, and PCV3, PCV4 plasmids (all at a concentration of 200.0 ng / μL) were used as templates, and standard plasmids pUC-DX5 and pUC-BT5, PRV-DX and PRV-BarthaK61 viral DNA were set as positive controls, and ddH2O was used as a negative control. All were diluted 10 3 The amplification was performed according to the reaction system and reaction conditions established in method example 2 to evaluate the specificity of the detection method of the present invention.

[0070] Specific test results such as Figure 5As shown in the figure, the detection system can only detect PRV genotype I and genotype II in the corresponding reaction channels, while no fluorescence signals can be detected in the reaction channels using PDCoV, CSFV, PRRSV, PEDV, PSFV, PCV2 virus, plasmids containing PCV3 and PCV4 full genomes as templates. This shows that the established three-probe fluorescence quantitative PCR method can distinguish PRV genotype I and genotype II, and does not cross-react with other common swine disease viruses, and has good specificity.

[0071] Example 4

[0072] Sensitivity test

[0073] 4.1 Establishment of standard curve

[0074] The constructed standard plasmids pUC-DX5 and pUC-BT5 were diluted with ddH2O to an initial copy number of 10 10 The standard plasmid was established by using a copy number of 10 copies / μL. 7 copies / μL up to 10 0 copies / μL, 2 μL of each dilution was taken as a template for fluorescent quantitative PCR reaction, and amplification was performed according to the established reaction conditions and reaction system to determine the minimum detection limit of the method established by the present invention and evaluate the sensitivity. The number of copies of each reaction corresponds to the cycle threshold value (Ct value) of the dilution, and the standard curve of PRV genotype I and genotype II is established.

[0075] Conversion formula between plasmid concentration (x: ng / μL) and copy number (y: copies / μL): y = (6.02×10 23 )×(x×10 -9 ) / (DNA length×660).

[0076] Take 10 1 copies / μL up to 10 6 The standard plasmids pUC-BT5 and pUC-DX5 with 10 copies / μL were used as templates, and the standard curves of PRV genotype I and genotype II were drawn according to the cycle threshold (Ct value) of each dilution concentration and the Log value of the corresponding copy number ( Figure 3 ).

[0077] The standard curve of PRV genotype I is Y=-3.492X+39.921 (Y: Ct value, X: logarithmic value of plasmid copy number), and the correlation coefficient is R 2 =0.9998.

[0078] The standard curve of PRV genotype II is Y = -3.5378X + 44.095, and the correlation coefficient is R2 =0.9998.

[0079] According to the standard curve, each reaction is 1 copies / μL~10 6 The linear relationship was good within the range of 2×10 1 copies / μL, with good sensitivity.

[0080] 4.2 Comparison of sensitivity between single-probe and multi-probe PCR detection methods

[0081] The single probe test was divided into two groups, one group changed the probe in the reaction system established in Example 2 to only the genotype I probe, the others were the same, and ddH2O was used to make up 20 μL of the system, and the other group changed the probe in the reaction system established in Example 2 to only the genotype II probe, the others were the same, and ddH2O was used to make up 20 μL of the system. The experimental method and reaction conditions were consistent with the standard curve, and the sensitivity of the multi-probe and single-probe PCR detection methods was compared.

[0082] The other conditions of the established detection system remain unchanged, the number of probes is reduced to a single probe, and the minimum detection limit is compared with the three-probe detection method established by the present invention. Figure 4 As shown in the figure, based on the detection system established by the present invention, the minimum detection limits of the single-probe and three-probe methods are both 2×10 1 copies / μL, indicating that the multi-probe method has a high detection sensitivity.

[0083] Example 5

[0084] Repeatability test

[0085] According to the established reaction conditions and reaction system, the constructed standard plasmids pUC-DX5 and pUC-BT5 were diluted 10-fold with ddH2O to a copy number ranging from 10 6 copies / μL up to 10 1 The results of the experiment are shown in Table 1. Each dilution was tested three times at different time points (1 day, 1 week, 1 month) under the same conditions to evaluate the inter-group repeatability of the real-time fluorescence quantitative PCR reaction. The test results show that the dilution from 10 6 copies / μL up to 10 1When the dilution rate was 200 copies / μL, the coefficient of variation of PRV genotype I as template was 1.52%, 1.62%, 1.47%, 1.19%, 0.94%, and 1.43%, and the coefficient of variation of PRV genotype II as template was 2.74%, 2.71%, 2.46%, 1.80%, 2.20%, and 1.91% (Table 2). The coefficient of variation of each dilution within and between groups was less than 3%, indicating that the method has good repeatability and can stably detect and identify PRV genotypes. The units of dilution concentrations in Tables 1 and 2 are copies·μL -1 .

[0086] Table 1 Results of repeated tests within the group

[0087]

[0088]

[0089] Table 2 Results of repeated tests between groups

[0090]

[0091]

[0092] Example 6

[0093] Clinical sample testing

[0094] After collecting clinical trial samples and extracting viral nucleic acid, the samples were tested using the triple fluorescence quantitative PCR typing method established in Example 2 and the PCR method published in the "Diagnostic Method for Pseudorabies" (GB / T 18641-2018), and the compliance rate with the PRV national standard detection method was compared to evaluate the clinical application value. At the same time, the reliability of this method was verified by sequencing and sequence analysis of the PCR products. The PCR amplification system and reaction conditions were the same as in Example 2.

[0095] The viral genomic DNA of 54 clinical samples was extracted as the template to be tested, and the three-probe fluorescent quantitative PCR detection method established in Example 2 was used for detection. PRV-DX and PRV-BarthaK61 viruses were used as positive controls, and ddH2O was used as a negative control. The results showed that there were 29 positive samples with a positive rate of 53.7%, which was higher than the 25.93% measured in the "Diagnostic Method for Pseudorabies" (GB / T18641-2018), and the method established by the present invention identified that the positive samples were highly consistent with the PRV gene type II sequence, with a consistency ratio of 99.64%. The products detected as positive were sequenced, and after comparison and analysis, the infected viruses were all PRV gene type II. The phylogenetic evolutionary tree results showed that these positive samples were all classified as PRV gene type II, which was consistent with the detection results of this method. It is shown that compared with the ordinary PCR method, the three-probe fluorescent quantitative PCR method established by the present invention can more sensitively detect positive clinical samples and has certain clinical application significance.

[0096] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A set of porcine pseudorabies virus genotyping probe primers, characterized in that: It comprises a pair of universal primers and a genotyping TaqMan probe, wherein the nucleotide sequence of the universal primer is shown as SEQ ID No.1 to SEQ ID No.2; The genotyping TaqMan probes include: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.

5.

2. The genotyping probe primer set according to claim 1, characterized in that: The 5' end of each genotyping TaqMan probe is labeled with a different fluorescent group, and the 3' end is labeled with a different quenching group.

3. The genotyping probe primer set according to claim 2, characterized in that: The fluorescent group is selected from TexasRed, 6-FAM or CY5; the quenching group is selected from BHQ2, MGB or BHQ3.

4. A detection kit for pseudorabies virus genotype I, characterized in that: It includes a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe is a type I probe, a type II probe and a universal probe; The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2; The nucleotide sequence of the type I probe is shown in SEQ ID No. 3; The nucleotide sequence of the type II probe is shown in SEQ ID No.4; The nucleotide sequence of the universal probe is shown in SEQ ID No.

5.

5. A detection kit for pseudorabies virus genotype II, characterized in that: It includes a pair of universal primers and a genotyping TaqMan probe; the genotyping TaqMan probe is a type I probe, a type II probe and a universal probe; The nucleotide sequences of the universal primers are shown in SEQ ID No.1 to SEQ ID No.2; The nucleotide sequence of the type I probe is shown in SEQ ID No. 3; The nucleotide sequence of the type II probe is shown in SEQ ID No.4; The nucleotide sequence of the universal probe is shown in SEQ ID No.

5.

6. A kit for genotyping of porcine pseudorabies virus, characterized in that: It includes a pair of universal primers and three genotyping TaqMan probes, wherein the nucleotide sequences of the universal primers are shown in SEQ ID No. 1 to SEQ ID No. 2; The genotyping TaqMan probes include: a type I probe with a nucleotide sequence as shown in SEQ ID No.3, a type II probe as shown in SEQ ID No.4, and a universal probe as shown in SEQ ID No.

5.

7. A method for genotyping porcine pseudorabies virus using a single probe for non-diagnostic purposes, characterized in that: The method comprises using the target genomic nucleic acid as a template, mixing with the detection kit according to claim 4 or the universal primers and genotyping TaqMan probes in claim 5 to configure a fluorescent quantitative PCR typing detection system, and after a PCR reaction, performing genotyping of the porcine pseudorabies virus according to the amplification result.

8. The method according to claim 7, characterized in that: The fluorescent quantitative PCR typing detection system is based on 20 μL, including: 10 μL of 2×AceQ qPCR Probe MasterMix, 2 μL of template, 0.9 μL of each universal primer, 0.4 μL of each genotyping TaqMan probe, and the balance of ddH2O.

9. The method according to claim 7, characterized in that: The PCR reaction procedure includes: 98°C for 5 min; 98°C for 10 sec, 60°C for 30 sec, and 40 cycles.

10. A method for genotyping porcine pseudorabies virus using multiple probes for non-diagnostic purposes, characterized in that: The method comprises using the target genomic nucleic acid as a template, mixing with the universal primers and genotyping TaqMan probes in the kit of claim 6 to configure a fluorescent quantitative PCR typing detection system, performing a PCR reaction, and then performing genotyping of the porcine pseudorabies virus according to the amplification result.

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