Primers and probes for specific detection of Cyprinid herpesvirus 2 and their applications

Abstract

The present invention relates to the technical field of biological virus detection, and specifically discloses primers and probes for specifically detecting Cyprinid herpesvirus 2 and their applications. Among the primers and probes for detecting Cyprinid herpesvirus 2, the sequence of the inner primer FIP is as shown in SEQ ID NO:1, the sequence of the inner primer BIP is as shown in SEQ ID NO:2, the sequence of the outer primer F3 is as shown in SEQ ID NO:3, the sequence of the outer primer B3 is as shown in SEQ ID NO:4, and the sequence of the loop primer probe is as shown in SEQ ID NO:5. By using the primers and probes of the present invention, the accurate detection of Cyprinid herpesvirus 2 can be achieved, the appearance of false positive results caused by non-specific amplification can be avoided, and the lowest detection limit can reach 0.5 copies / μL, which is more conducive to the early detection of Cyprinid herpesvirus 2, thereby avoiding the spread of diseases, and has very important significance for the prevention and control of aquatic diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biological virus detection, and particularly to a primer and a probe for specifically detecting Cyprinid edematosis virus and their applications. Background Art

[0002] Carp is the most widely cultured fish globally. Cyprinid edematosis, koi herpesvirus disease, and spring viraemia of carp are the main current viral diseases, causing significant economic losses to the aquaculture industries in various countries around the world.

[0003] Currently, there is relatively little research on the detection of Cyprinid edematosis virus at home and abroad. The main detection methods for CEV are fluorescence PCR method, nested PCR method, agarose electrophoresis method, LAMP method, etc. However, the existing detection methods still have obvious defects such as complex operation, long detection time, and easy generation of false positive results due to non-specific amplification, which greatly limits the application and popularization of these methods in the detection of Cyprinid edematosis virus. Therefore, developing a rapid detection method for Cyprinid edematosis virus that is simple, highly specific, and avoids false positive detection is of great significance for the early prevention and treatment of Cyprinid edematosis virus. Summary of the Invention

[0004] Aiming at the problems of high false positive probability, long detection time, and low sensitivity existing in the existing detection methods for Cyprinid edematosis virus, the present invention provides a primer and a probe for specifically detecting Cyprinid edematosis virus and their applications. Using the primer and probe for detecting Cyprinid edematosis virus, Cyprinid edematosis virus can be rapidly detected under isothermal conditions, and the occurrence of false positive detection results can be completely avoided. It has good specificity and high sensitivity, which is beneficial to realizing the early prevention and treatment of Cyprinid edematosis virus, thereby effectively preventing the spread of the disease and reducing the economic losses of farmers.

[0005] To achieve the above invention purpose, the embodiments of the present invention adopt the following technical solutions:

[0006] A primer and a probe for specifically detecting Cyprinid edematosis virus, including:

[0007] Inner primer FIP: 5′-GTTCATTCTCAAATGGTTGCAACACTGCCATTACGTAATTAGAATCG-3′ (SEQ ID NO:1);

[0008] Inner primer BIP: 5′-AGTTGATATGCTTTTGCATTTGCAAGAAGCTCTACTTTGTCTCA-3′ (SEQ ID NO:2);

[0009] Outer primer F3: 5′-ATGGATTTGCTGCTGGTG-3′ (SEQ ID NO:3);

[0010] Outer primer B3: 5′-GCTTCTCAAGATATACATTACACA-3′ (SEQ ID NO:4);

[0011] Loop primer probe LBP: 5′-GCAACAACTTGACGAGGGAATGAT-3′ (SEQ ID NO:5).

[0012] Compared with the prior art, the primers and probes for specifically detecting Cyprinid herpesvirus 2 provided by the present invention can specifically bind to the nucleic acid of Cyprinid herpesvirus 2, accurately detect Cyprinid herpesvirus 2, and by designing a loop primer and designing the loop primer as the loop primer probe LBP, not only is non-specific amplification reduced, avoiding the occurrence of false positive detection results, but also the detection sensitivity of Cyprinid herpesvirus 2 is improved, and the lowest detection limit of Cyprinid herpesvirus 2 can reach 0.5 copies / μL. The primers and probes provided by the present invention have no cross-amplification reaction with 16 viruses such as Goldfish hematopoietic necrosis virus, Spring viraemia of carp virus, Grass carp reovirus, Cyprinid herpesvirus 1, Cyprinid herpesvirus 2, Cyprinid herpesvirus 3, etc., and the specificity is significantly enhanced; at the same time, the detection efficiency is significantly improved, rapid amplification is carried out under the constant temperature condition of 63°C, and the detection level can be reached only in 50 min, the detection time is significantly shortened, which has important significance for the prevention and control of Cyprinid herpesvirus 2 diseases, can effectively reduce the economic losses of farmers, and has a broad application market prospect in the precise and rapid detection of aquatic diseases.

[0013] Preferably, the loop primer probe LBP is: FAM-5′-GCAACAACTTGACGAGGGAATGAT-3′BHQ.

[0014] The present invention also provides a kit for specifically detecting Cyprinid herpesvirus 2, comprising the primers and probes described in any one of the above.

[0015] Preferably, it includes a pre-reaction solution, a positive control solution and a negative control solution, wherein the pre-reaction solution includes a basic buffer solution, a potassium chloride solution, an ammonium sulfate solution, Triton X-100, a DNA polymerase, betaine, a magnesium chloride solution, dTNPs and deionized water.

[0016] More preferably, the basic buffer solution is Tris-HCl.

[0017] More preferably, the basic buffer solution is Tris-HCl with a pH of 8.3.

[0018] More preferably, the DNA polymerase includes Bst DNA polymerase and Taq DNA polymerase.

[0019] Preferably, the negative control solution is ddH2O.

[0020] Preferably, the concentration of the positive control solution is 5×10 4 copies / μL.

[0021] It should be noted that the positive control solution is prepared by diluting a standard plasmid. The standard plasmid is designed and constructed based on the conserved region of the whole gene sequence of Cyprinid herpesvirus 2. An engineering bacterium containing a recombinant plasmid is synthesized by Shanghai Sangon Biotech Co., Ltd. After fermentation, plasmid extraction is carried out with reference to the plasmid miniprep kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the plasmid concentration and purity are determined using a NanoDrop One / OneC Microvolume UV-Vis Spectrophotometer (ND-100C, MIULAB).

[0022] The present invention also provides a method for detecting Cyprinid herpesvirus 2 using the above-mentioned kit. The specific operation is as follows: Extract the nucleic acid of Cyprinid herpesvirus 2 as a template, perform PCR amplification using the kit, and perform real-time fluorescence detection during the amplification process.

[0023] If both the test sample and the positive control can amplify a fluorescence signal, while the negative control cannot amplify a fluorescence signal, it indicates that the test sample is positive for Cyprinid herpesvirus 2; if neither the test sample nor the negative control can amplify a fluorescence signal, while the positive control can amplify a fluorescence signal, it indicates that the test sample is negative for Cyprinid herpesvirus 2.

[0024] Preferably, the PCR amplification system includes the following reagents and amounts: 20 mmol of pH 8.3 Tris-HCl, 10 mmol of KCl, 10 mmol of (NH4)2SO4, 0.025 μL of Triton X-100, 8 U of Bst DNA polymerase, 2.5 U of Taq DNA polymerase, 1 mol of betaine, 8 mmol of MgCl2, 2.0 mmol of dNTPs, 2.0 μmol of inner primer FIP, 2.0 μmol of inner primer BIP, 0.25 μmol of outer primer F3, 0.25 μmol of outer primer B3, 0.25 μmol of loop primer probe LBP, and deionized water is added to make up to 23 μL.

[0025] The above-mentioned preferred reaction conditions can further improve the detection efficiency of Cyprinid herpesvirus 2.

[0026] Preferably, the reaction temperature for PCR amplification is 62°C - 64°C, and the reaction time is 50 min - 55 min.

[0027] More preferably, the reaction temperature for PCR amplification is 63°C, and the reaction time is 50 min.

[0028] The preferred PCR amplification conditions can improve the detection efficiency of Cyprinid herpesvirus 2.

[0029] The detection method of Cyprinid edematosis virus provided by the present invention is simple to operate. The detection result can be determined by analyzing the real-time fluorescence quantitative PCR amplification curve, without the need to observe by gel electrophoresis method. The analysis result is simple and clear, and effectively avoids the occurrence of false positive detection results. The detection limit can reach 0.5 copies / μL, which is more conducive to realizing the early detection of Cyprinid edematosis virus, early discovery of diseases, and effective prevention of the spread of diseases, and has broad application prospects. Brief Description of the Drawings

[0030] Figure 1 It is the structural diagram of the standard quality plasmid in Example 1 of the present invention;

[0031] Figure 2 It is the specific test result diagram of the Cyprinid edematosis virus detection method in Example 2 of the present invention;

[0032] Figure 3 It is the sensitivity test result diagram of the Cyprinid edematosis virus detection method in Example 2 of the present invention; among them, the amplification curves are in sequence from left to right as 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 50 copies / μL, 5 copies / μL and 0.5 copies / μL;

[0033] Figure 4 It is the repeatability test result diagram of the Cyprinid edematosis virus detection method in Example 2 of the present invention. Detailed Embodiments

[0034] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] To better illustrate what is provided in the embodiments of the present invention, further illustrative examples will be given below through embodiments.

[0036] Example 1

[0037] 1. Materials and Methods

[0038] 1.1 Virus Samples and Kits

[0039] The carp edema virus (CEV), goldfish hematopoietic necrosis virus (GFHNV), spring viremia of carp virus (SVCV), grass carp reovirus (GCRV), cyprinid herpesvirus 1 (CyHV-1), cyprinid herpesvirus 2 (CyHV-2), cyprinid herpesvirus 3 (CyHV-3), epizootic hematopoietic necrosis virus (EHNV), herpesvirus of anguillid (HVA), channel catfish virus disease virus (CCVD), infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus (VHSV), tilapia lake virus (TiLV), giant salamander iridovirus (GSIV), Bohle iridovirus (BIV), white spot syndrome virus of shrimp (WSSV), and Enterocytozoon hepatopenaei (EHP) were all isolated, identified by clinical detection in our laboratory and then preserved.

[0040] 1.2 Design of primers and probes

[0041] Primers and probes were designed based on the conserved sequence of the carp edema virus specific gene (Sequence ID: MF326541.1). The designed primer sequences are as follows and were synthesized and labeled by Shanghai Sangon Biotech Co., Ltd.

[0042] Inner primer FIP: 5′-GTTCATTCTCAAATGGTTGCAACACTGCCATTACGTAATTAGAATCG-3′ (SEQ ID NO: 1);

[0043] Inner primer BIP: 5′-AGTTGATATGCTTTTGCATTTGCAAGAAGCTCTACTTTGTCTCA-3′ (SEQ ID NO: 2);

[0044] Outer primer F3: 5′-ATGGATTTGCTGCTGGTG-3′ (SEQ ID NO: 3);

[0045] Outer primer B3: 5′-GCTTCTCAAGATATACATTACACA-3′ (SEQ ID NO: 4);

[0046] Loop primer probe LBP: FAM-5′-GCAACAACTTGACGAGGGAATGAT-3′BHQ.

[0047] 1.3 Extraction of DNA / RNA

[0048] DNA of carp edema virus, goldfish hematopoietic necrosis virus, cyprinid herpesvirus 1, cyprinid herpesvirus 2, cyprinid herpesvirus 3, herpesvirus of anguillid, channel catfish virus disease virus, giant salamander iridovirus, and white spot syndrome virus of shrimp was extracted respectively according to the instruction manuals of commercially available DNA extraction kits.

[0049] Extract the RNA of spring viraemia of carp virus, grass carp reovirus, epizootic haematopoietic necrosis virus, infectious haematopoietic necrosis virus, viral haemorrhagic septicaemia virus, tilapia lake virus, Bohle virus, and Enterocytozoon hepatopenaei respectively according to the instructions of commercially available RNA extraction kits.

[0050] Store the extracted DNA and RNA at -20 °C for subsequent experiments.

[0051] 1.4 Construction of standard quality plasmid

[0052] Recombine the conserved sequence of the koi herpesvirus genome (Sequence ID: MF326541.1) into the PUC57 plasmid to obtain a recombinant plasmid, as Figure 1 shown. Prepare the engineering bacteria containing the recombinant plasmid by Shanghai Sangon Biotech. After fermentation, extract the plasmid with reference to the plasmid miniprep kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). Determine the concentration and purity of the plasmid using a NanoDrop One / OneC Microvolume UV-Vis Spectrophotometer (ND-100C, MIULAB). The concentration of the standard quality plasmid is 5×10 7 copies / μL.

[0053] 1.5 Establishment of a method for detecting koi herpesvirus

[0054] The kit for detecting koi herpesvirus includes a pre-reaction solution, a positive control solution, and a negative control solution. Among them, the pre-reaction solution includes a basic buffer solution, potassium chloride solution, ammonium sulfate solution, Triton X-100, DNA polymerase, betaine, magnesium chloride solution, dTNPs, and deionized water. Among them, the negative control is ddH2O, and the positive control solution is prepared by diluting the standard plasmid, with a concentration of 5×10 4 copies / μL.

[0055] Use the nucleic acid extracted from koi herpesvirus as a template, and combine the above kit to construct a PCR amplification reaction system (23 μL): 20 mmol pH8.3 Tris-HCl, 10 mmol KCl, 10 mmol (NH 4)2 SO4, 0.025 μL Triton X-100, 8 U Bst DNA polymerase, 2.5 U Taq DNA polymerase, 1 mol betaine, 8 mmol MgCl2, 2.0 mmol dNTPs, 2.0 μmol inner primer FIP, 2.0 μmol inner primer BIP, 0.25 μmol outer primer F3, 0.25 μmol outer primer B3, 0.25 μmol loop primer probe LBP, and make up to 23 μL with deionized water.

[0056] Add 2 μL of the nucleic acid template to be tested to a reaction tube containing 23 μL of the above reaction solution, perform real-time fluorescence quantitative PCR amplification detection, incubate at a constant temperature of 63 °C for 50 min, and analyze the amplification results.

[0057] If fluorescence signals can be amplified from both the sample to be tested and the positive control, while no fluorescence signal can be amplified from the negative control, it indicates that the sample to be tested is positive for Cyprinid herpesvirus 2; if no fluorescence signals can be amplified from both the sample to be tested and the negative control, while a fluorescence signal can be amplified from the positive control, it indicates that the sample to be tested is negative for Cyprinid herpesvirus 2.

[0058] Example 2

[0059] 2.1 Specificity test for detecting Cyprinid herpesvirus 2:

[0060] Extract the DNA of Cyprinid herpesvirus 2, Goldfish hematopoietic necrosis virus, Cyprinid herpesvirus 1, Cyprinid herpesvirus 2, Cyprinid herpesvirus 3, Eel herpesvirus, Channel catfish virus, Giant salamander iridovirus, White spot syndrome virus of shrimp; and the RNA of Spring viraemia of carp virus, Grass carp reovirus, Epizootic haematopoietic necrosis virus, Infectious haematopoietic necrosis virus, Viral haemorrhagic septicaemia virus, Tilapia lake virus, Bohle iridovirus, Enterocytozoon hepatopenaei respectively according to the instructions of commercially available DNA / RNA extraction kits as samples to be tested.

[0061] It is optional to use the ME02B08 nucleic acid extraction kit of Hebei Sanshi Biotechnology Co., Ltd. to extract DNA / RNA.

[0062] PCR amplification system: 20 mmol of Tris-HCl with pH 8.3, 10 mmol of KCl, 10 mmol of (NH 4)2 SO4, 0.025 μL of TritonX-100, 8 U of Bst DNA polymerase, 2.5 U of Taq DNA polymerase, 1 mol of betaine, 8 mmol of MgCl2, 2.0 mmol of dNTPs, 2.0 μmol of inner primer FIP, 2.0 μmol of inner primer BIP, 0.25 μmol of outer primer F3, 0.25 μmol of outer primer B3, 0.25 μmol of loop primer probe LBP, and make up to 23 μL with deionized water. Use ddH2O as the negative control and the dilution of the standard quality plasmid as the positive control, with a concentration of 5×10 4 copies / μL.

[0063] It should be noted that if the sample to be tested is an RNA virus, add 8 U of StartScript Reverse Tarnscriptase to the above amplification system, and other conditions remain unchanged.

[0064] The detection results are as follows Figure 2 shown. As can be seen from the figure, only the Cyprinid herpesvirus 2 shows a typical S-shaped amplification curve, while no amplification occurs for the nucleic acids of other viruses, demonstrating that the Cyprinid herpesvirus 2 detection system provided by the present invention has good specificity.

[0065] 2.2 Sensitivity test for detecting Cyprinid herpesvirus 2:

[0066] The standard quality plasmid (plasmid concentration of 5×10 7 copies / μL) was diluted 10-fold (5×10 6 copies / μL) as the starting concentration for the test.

[0067] The test plasmid of 5×10 6 copies / μL was successively diluted 10-fold in 7 gradients to 0.5 copies / μL, that is, the concentrations of the test plasmid were 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL, 50 copies / μL, 5 copies / μL, and 0.5 copies / μL. The test plasmids at the above 8 gradient concentrations were used as templates for systematic testing, and the empty vector PUC57 plasmid was used as a negative control. The detection results are as follows Figure 3 shown.

[0068] The results show that the lowest detection limit concentration of the method for detecting Cyprinid herpesvirus 2 provided by the present invention is 0.5 copies / μL (0.286 fg / μL), the correlation coefficient R 2 is 0.995, and there is no non-specific amplification in the negative control.

[0069] 2.3 Repeatability verification of the method for detecting Cyprinid herpesvirus 2:

[0070] The test plasmid with a concentration of 5×10 4 copies / μL was used as the repeatability detection sample, and 10 repetitions were tested. Detection was carried out by the detection method for Cyprinid herpesvirus 2 established by the present invention. The results are as follows Figure 4 shown.

[0071] The test results show that the coefficient of variation Cv value of the repeatability detection is 1.52%, which is less than 3%, indicating that the repeatability of the specific method for detecting Cyprinid herpesvirus 2 provided by the present invention is good.

[0072] Example 3

[0073] Clinical sample detection:

[0074] Using the fluorescence PCR method of the aquatic industry standard SC / T 7229-2019 as the control method, 60 carp samples collected were tested in parallel according to the koi edema virus detection method provided by the present invention. The established detection accuracy rate was 100% (60 / 60), the sensitivity was 100% (19 / 19), and the specificity was 100% (41 / 41). The results are shown in Table 1.

[0075] Table 1

[0076]

[0077] Comparative Example 1

[0078] The PCR method and LAMP method in the aquatic industry standard SC / T 7229-2019 were compared with the koi edema virus detection method provided by the present invention. And nucleic acids of the same early-stage diseased carp sample were extracted by different extraction methods to compare the stability of different detection methods.

[0079] The nucleic acid extraction methods included rapid extraction method, extraction method, column extraction method, and magnetic bead hand-held method. Among them, the extraction method was the extraction method in the aquatic industry standard SC / T 7229-2019, the column extraction method used the DP201 DNA / RNA extraction kit (column extraction method) of Hebei Sanshi Biotechnology Co., Ltd., the magnetic bead hand-held method used the DP301 nucleic acid extraction kit (magnetic bead method) of Hebei Sanshi Biotechnology Co., Ltd., and the rapid extraction method used the ME02B08 nucleic acid extraction kit of Hebei Sanshi Biotechnology Co., Ltd. Nucleic acids extracted by different methods were all prepared into the same concentration (0.5 ng / μL).

[0080] Then, the PCR method and LAMP method in SC / T 7229-2019 and the koi edema virus detection method provided by the present invention were respectively used to perform real-time fluorescence quantitative PCR amplification detection on the extracted nucleic acids. The results are shown in Table 2.

[0081] Table 2

[0082]

[0083]

[0084] The types and contents of impurity residues in nucleic acid samples extracted by different nucleic acid extraction methods are different, and the degree of influence on the specific amplification of nucleic acid is also different. By comparing four nucleic acid extraction methods and performing fluorescence quantitative detection through three different detection methods, it can be seen from the detection results that for the nucleic acid of the initial diseased fish samples, due to the low content of Cyprinid herpesvirus 2, there is little difference in the detection time between the negative control and the sample by the LAMP method, and it is impossible to judge whether it is a positive sample of Cyprinid herpesvirus 2.

[0085] The detection method for specific Cyprinid herpesvirus 2 provided by the present invention is applicable to all four different nucleic acid extraction methods. There is a false positive problem in the LAMP method during the detection process. Although the PCR method has strong specificity, its amplification is slow and the Ct value is large.

[0086] In summary, the detection method for specific Cyprinid herpesvirus 2 provided by the present invention is not only simple, rapid, but also has strong applicability and good specificity. It can effectively improve the occurrence of false positive problems in detection, is more conducive to the early detection of Cyprinid herpesvirus 2, and thus avoids the spread of diseases, which has very important significance for the prevention and control of aquatic diseases.

[0087] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer and a probe for specifically detecting Cyprinid herpesvirus 2, characterized in that, Comprising: Inner primer FIP: 5′-GTTCATTCTCAAATGGTTGCAACACTGCCATTACGTAA TTAGAATCG-3′; Inner primer BIP: 5′-AGTTGATATGCTTTTGCATTTGCAAGAAGCTCTACTTT GTCTCA-3′; Outer primer F3: 5′-ATGGATTTGCTGCTGGTG-3′; Outer primer B3: 5′-GCTTCTCAAGATATACATTACACA-3′; Loop primer probe LBP: 5′-GCAACAACTTGACGAGGGAATGAT-3′.

2. The primer and probe for specifically detecting Cyprinid herpesvirus 2 according to claim 1, characterized in that, The loop primer probe LBP is: FAM-5′-GCAACAACTTGACGAGGGAATGAT-3′BHQ.

3. A kit for specifically detecting Cyprinid herpesvirus 2, characterized in that, Comprising the primer and probe according to any one of claims 1-2.

4. The kit for specifically detecting Cyprinid herpesvirus 2 according to claim 3, characterized in that, Including a pre-reaction solution, a positive control solution and a negative control solution, wherein the pre-reaction solution includes a basic buffer solution, a potassium chloride solution, an ammonium sulfate solution, Triton X-100, a DNA polymerase, betaine, a magnesium chloride solution, dTNPs and deionized water.

5. The kit for specifically detecting Cyprinid herpesvirus 2 according to claim 4, wherein The negative control solution is ddH2O.

6. The kit for specifically detecting Cyprinid herpesvirus 2 according to claim 4, wherein, The concentration of the positive control solution is 5×10 4 copies / μL.

7. A method for detecting Cyprinid herpesvirus 2 using the kit according to claim 3 for non-diagnostic purposes, characterized in that, The specific operation is: using the nucleic acid extracted from the carp edema virus as a template, and performing real-time fluorescence quantitative PCR amplification detection with the kit.

8. The method for detecting Cyprinid herpesvirus 2 for non-diagnostic purposes according to claim 7, characterized in that: The PCR amplification system includes the following reagents and dosages: 20 mmol of Tris-HCl with pH 8.3, 10 mmol of KCl, 10 mmol of (NH 4)2 SO4, 0.025 μL of Triton X-100, 8 U of Bst DNA polymerase, 2.5 U of Taq DNA polymerase, 1 mol of betaine, 8 mmol of MgCl2, 2.0 mmol of dNTPs, 2.0 μmol of inner primer FIP, 2.0 μmol of inner primer BIP, 0.25 μmol of outer primer F3, 0.25 μmol of outer primer B3, 0.25 μmol of loop primer probe LBP, and deionized water is added to make up to 23 μL.

9. The method for detecting Cyprinid herpesvirus 2 for non-diagnostic purposes according to claim 7, characterized in that: The reaction temperature for PCR amplification is 62°C - 64°C, and the reaction time is 50 min - 55 min.

10. The method for detecting Cyprinid herpesvirus 2 for non-diagnostic purposes according to claim 9, characterized in that: The reaction temperature for PCR amplification is 63°C, and the reaction time is 50 min.

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