Universal porcine reproductive and respiratory syndrome virus detection primer, probe and kit

Abstract

The invention discloses a universal porcine reproductive and respiratory syndrome virus detection primer, a probe and a kit, and relates to the technical field of virus detection, in particular to a universal porcine reproductive and respiratory syndrome virus detection primer and probe composition and a kit. The universal porcine reproductive and respiratory syndrome virus detection primers are ORF6-F and ORF6-R, and the detection probe is ORF6-P. The kit disclosed by the invention has the characteristics of high detection sensitivity, high detection specificity and high detection stability on the gene type I and the gene type II of the porcine reproductive and respiratory syndrome virus, and belongs to a universal detection reagent for the porcine reproductive and respiratory syndrome virus. The kit is suitable for large-scale detection work in pig farms and the like, and has the remarkable advantages of high detection rate, short detection time and the like; and for complex samples, such as serum, nasopharynx swabs, tissues and the like, an excellent detection rate is shown.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a universal primer and probe composition and kit for detecting porcine reproductive and respiratory syndrome virus. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a highly contagious disease in pigs caused by the porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV can infect pigs of different breeds, sexes, and ages, leading to immunosuppression. Its main symptoms include stillbirth, abortion, mummified fetuses, weak piglets, and respiratory diseases. PRRSV is a single-stranded positive-sense RNA virus; the viral particles are oval and enveloped, and it is classified in the order Heliovirales, family Arterioviridae, and genus Arteriovir. PRRSV is divided into two genotypes: the European type (genotype I), represented by LV, and the American type (genotype II), represented by VR-2332.

[0003] The PRRSV genome is approximately 15 kb in length and contains 10 open reading frames (ORFs). Among them, ORF1a and ORF1b are the RNA replicase coding regions necessary for viral replication, which, after processing, can be cleaved to produce at least 12 smaller non-structural proteins (nsp). In addition, ORF2a, ORF2b, ORF3, ORF4, ORF5a, ORF5, ORF6, and ORF7 encode the virus's structural proteins.

[0004] Pigs are the only natural host for PRRSV virus, and they become infected and develop clinical symptoms after contact with the virus. Whether pigs develop clinical symptoms after infection or recover after treatment, they can continue to shed the virus for a long period. Therefore, accurate diagnosis and detection are crucial prerequisites for effectively controlling the spread of PRRSV virus. Because PRRSV constantly undergoes new mutations, deletions, and recombinations to produce new genotypes, it has become the disease with the highest mutation and variation rate in the field of swine diseases. Existing detection methods each have their advantages and disadvantages:

[0005] Virus isolation is one of the most accurate methods for diagnosing PRRSV. It involves using PAM cells or MAC145 cells as host culture cells and observing the cell culture status to determine whether tissue is infected with PRRSV. However, tissue samples from which the virus is isolated need to be stored under suitable temperature and pH conditions. Furthermore, pig farms are often located in remote areas, far from residential areas, leading to numerous difficulties in the transportation and preservation of viral tissues. In addition, the long virus isolation period hinders timely prevention and monitoring on pig farms.

[0006] ELISA is a highly sensitive and specific method with standardized procedures and result interpretation, making it suitable for large-scale sample testing. However, for samples with trace amounts or high levels of impurities, the specificity and sensitivity of ELISA can significantly decrease, easily leading to missed or false diagnoses.

[0007] Conventional RT-PCR is a commonly used detection method in pig farms, but it has low sensitivity, the electrophoresis process is harmful to humans, and the operation is relatively complex. In contrast, quantitative real-time RT-PCR is not only highly sensitive, specific, and reproducible, but also provides intuitive results. Most importantly, it has a short detection time, which can significantly shorten the detection time for diseased pig farms. It should be the most ideal detection method for porcine reproductive and respiratory syndrome (PRRS), but existing quantitative real-time RT-PCR methods still have many shortcomings.

[0008] Chinese invention patent application CN202310122481.2 discloses a universal primer composition, kit, and detection method for detecting porcine reproductive and respiratory syndrome virus (PRRSV). However, verification experiments were conducted based on the disclosed primer pairs, and the experimental results are as follows: Figure 1 As shown, it was found that the downstream primer in its universal primer composition did not match type I porcine reproductive and respiratory syndrome virus (PRRSV), and could not accurately detect both genotypes of PRRSV.

[0009] IDEXX's commercially available "Porcine Reproductive and Respiratory Syndrome Type 1-2 Multiplex Real-Time Quantitative PCR Detection Kit" provides accurate PRRSV detection results; however, this kit uses separate primers and probes for type 1 (European strain) and type 2 (American strain), requiring separate detection through three fluorescence channels. For laboratory fluorescence quantitative instruments with only one channel, or for large-scale detection, this results in excessively high detection costs and a cumbersome detection process, which is not conducive to the actual detection needs of large-scale breeding units such as pig farms. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing PRRSV fluorescence quantitative RT-PCR and to provide a universal primer and probe composition and kit for detecting porcine reproductive and respiratory syndrome virus.

[0011] This invention relates to universal primers and probes for detecting porcine reproductive and respiratory syndrome virus (PRRSV), wherein the universal primers for detecting PRSV are ORF6-F and ORF6-R, and the detection probe is ORF6-P.

[0012] The ORF6-F sequence is: 5'-TGCCGKTTGTGCTTGCTAG-3';

[0013] The ORF6-R sequence is: 5'-GGACGACAAATGCGTGGTTATC-3';

[0014] The ORF6-P sequence is: 5'-CGCAAGTACATTCTGG-3'; the 5' end of the detection probe ORF6-P sequence carries a fluorescent reporter group, and the 3' end carries a fluorescent quencher group.

[0015] Furthermore, the 5' end fluorescent reporter group of the detection probe sequence is FAM, and the 3' end fluorescent quencher group is MGB.

[0016] This invention discloses a real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus (PRRSV), comprising universal PRSV detection primers ORF6-F and ORF6-R and detection probe ORF6-P.

[0017] Furthermore, the kit also includes a one-step RNase system, comprising 5×one step u+ mix and one step u+ enzyme mix.

[0018] The above-mentioned real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus (PRRSV) is used in the preparation of products for diagnosing PRRSV.

[0019] Application of the above-mentioned universal primers for detecting porcine reproductive and respiratory syndrome virus (PRRSV) in the preparation of a real-time fluorescent RT-PCR detection kit for PRSV.

[0020] The application of the above-mentioned universal porcine reproductive and respiratory syndrome virus (PRRSV) detection primers and probes in the preparation of a real-time fluorescent RT-PCR detection kit for PRSV.

[0021] This invention discloses a method for identifying porcine reproductive and respiratory syndrome virus (PRRSV) for non-disease diagnostic purposes, comprising the following steps:

[0022] Step 1: Preparation of the real-time RT-PCR reaction system and pre-reaction procedures;

[0023] Step 2: Nucleic acid extraction from the test sample;

[0024] Step 3: Quantitative Real-Time RT-PCR procedure;

[0025] Step 4: The real-time PCR instrument reads the CT value of the test sample. If the CT value is <35, the test sample is considered positive. If the CT value is between 35 and 40, the sample is considered suspicious and nucleic acid needs to be extracted again for real-time RT-PCR test. If the CT value of the second amplification is still between 35 and 40, it is considered positive.

[0026] The virus detection primers in the real-time RT-PCR reaction system are ORF6-F and ORF6-R, and the detection probe is ORF6-P.

[0027] Furthermore, the reaction procedure for step three, quantitative RT-PCR, is as follows: first stage reverse transcription: 55℃ for 15 min; second stage pre-denaturation: 95℃ for 30 s; third stage cycling: 95℃ for 10 s, 60℃ for 30 s, for a total of 45 cycles; fluorescence signal is collected at 60℃, using the FAM fluorescence channel.

[0028] Further, in step one, the reaction system is prepared as follows: take out the one-step RNA enzyme system, virus detection primers ORF6-F and ORF6-R, detection probe ORF6-P, positive control, and negative control, place them in a 4°C refrigerator to thaw completely, briefly and thoroughly shake them, then centrifuge them briefly, and then prepare the PCR reaction solution.

[0029] Furthermore, the one-step RNase system consisted of 5× one-step u+ mix and one-step u+ enzyme mix (purchased from Nanjing Novizan Biotechnology Co., Ltd.), with a positive control being porcine reproductive and respiratory syndrome virus plasmid and a negative control being ddH2O.

[0030] Furthermore, if the sample to be tested in step two is tissue, the tissue should be ground first, and then centrifuged to obtain the supernatant for later use; if the sample to be tested is serum, nasopharyngeal swab, environmental sample or testicular fluid, nucleic acid can be extracted directly using a fully automated DNA / RNA nucleic acid extraction kit.

[0031] The detection of porcine reproductive and respiratory syndrome virus using the universal primers and probes of this invention has the advantages of high specificity, accurate quantitative detection, and high sensitivity.

[0032] This invention's kit exhibits high detection sensitivity, specificity, and stability for both porcine reproductive and respiratory syndrome virus (PRRSV) genotypes I and II, making it a universal detection reagent for PRSV. The kit is suitable for large-scale testing in pig farms and other similar facilities, offering significant advantages such as high detection rates and short testing times. It also demonstrates excellent detection rates for complex samples, including serum, nasopharyngeal swabs, and tissues. Attached Figure Description

[0033] Figure 1 The figure shows the results of a verification experiment on porcine reproductive and respiratory syndrome virus (PRRSV) genotype I and genotype II based on the universal detection primer composition, kit and detection method disclosed in the prior art CN202310122481.2.

[0034] Figure 2 This is a gel electrophoresis result image of Example 2;

[0035] Figure 3 This is a comparison diagram of the amplification results of typical genotype I and genotype II porcine reproductive and respiratory syndrome virus strains using the universal porcine reproductive and respiratory syndrome virus detection primers ORF6-F and ORF6-R of the present invention in Example 2.

[0036] Figure 4 This is a graph showing the primer specificity test results from Example 3;

[0037] Figure 5 This is the amplification curve of the porcine reproductive and respiratory syndrome virus obtained in Example 4;

[0038] Figure 6 This is a standard curve of the test kit obtained in Example 4;

[0039] Figure 7 This is a graph showing the sensitivity test results of the test kit in Example 5;

[0040] Figure 8 This is a graph showing the test results of the sample verification experiment in Example 6;

[0041] Figure 9 This is a bar chart showing the detection volume of porcine reproductive and respiratory syndrome virus using the kit of this invention from 2023 to August 2025.

[0042] Figure 10 This refers to the number of samples tested for porcine reproductive and respiratory syndrome virus in the Heilongjiang region using the kit of this invention from 2024 to August 2025. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0045] Example 1: This example illustrates a method for identifying porcine reproductive and respiratory syndrome virus (PRRSV) for non-disease diagnostic purposes, comprising the following steps:

[0046] Step 1: Preparation of the reaction system and pre-reaction procedures;

[0047] Step 2: Nucleic acid extraction from the test sample;

[0048] Step 3: Quantitative Real-Time RT-PCR procedure;

[0049] Step 4: The real-time PCR instrument reads the CT value of the test sample. If the CT value is <35, the test sample is considered positive. If the CT value is between 35 and 40, the sample is considered suspicious and nucleic acid needs to be extracted again for real-time RT-PCR test. If the CT value of the second amplification is still between 35 and 40, it is considered positive.

[0050] The virus detection primers in the real-time RT-PCR reaction system are ORF6-F and ORF6-R, and the detection probe is ORF6-P.

[0051] The reaction procedure for step three, quantitative RT-PCR, is as follows: first stage reverse transcription: 55℃ for 15 min; second stage pre-denaturation: 95℃ for 30 s; third stage cycling: 95℃ for 10 s, 60℃ for 30 s, for a total of 45 cycles; fluorescence signal is collected at 60℃, and the FAM fluorescence channel is selected.

[0052] In step one, the reaction system is prepared as follows: Take the one-step RNase system, virus detection primers ORF6-F and ORF6-R, detection probe ORF6-P, positive control, and negative control, and place them in a 4°C refrigerator to completely thaw. After brief and thorough shaking, perform a short-term centrifugation to prepare the PCR reaction solution. A 15 μL PCR reaction solution is prepared by combining 4 μL of one-step RNase system, 1 μL of one-tube reverse transcription premix, 1.5 μL of primers ORF6-F and ORF6-R, 1 μL of detection probe ORF6-P, and the remainder ddH2O.

[0053] The one-step RNase system consisted of 5×one step u+ mix and one step u+ enzyme mix (purchased from Nanjing Novizan Biotechnology Co., Ltd.), with a positive control being porcine reproductive and respiratory syndrome virus plasmid and a negative control being ddH2O.

[0054] In step two, if the sample is tissue, the tissue should be ground first, and then centrifuged to obtain the supernatant for later use. If the sample is serum, nasopharyngeal swab, environmental sample or testicular fluid, nucleic acid can be extracted directly using a fully automated DNA / RNA nucleic acid extraction kit.

[0055] In the real-time RT-PCR reaction system, the concentration range of upstream and downstream primers and probes for virus detection is 1~10 μmol / L.

[0056] In step one, the corresponding PCR reaction solution is prepared according to the number of samples to be tested and dispensed into PCR eight-tube strips. In step three, the samples to be tested, negative controls, and positive controls are added in sequence. After the tube caps are tightened, the PCR eight-tube strips are shaken to mix and then briefly centrifuged. The briefly centrifuged PCR eight-tube strips are then placed in a real-time PCR instrument and the FAM fluorescence channel is selected.

[0057] In step three, the ratio of PCR reaction solution to nucleic acid in the test sample is 15ul + 5ul; the amount of positive control and negative control added is the same as the amount of nucleic acid in the test sample.

[0058] The required number of reaction tubes is calculated according to the one-step RNase preparation method, with a ratio of 15 × (n + 1), and thorough mixing is ensured. Here, n represents the number of preparation systems.

[0059] In this embodiment, the corresponding CT value is read using a real-time PCR instrument. If the fluorescent group FAM shows a typical "S"-shaped amplification curve, it can be determined to be positive for porcine reproductive and respiratory syndrome virus; if no amplification curve appears, it is determined to be negative.

[0060] Example 2

[0061] Validation experiment of universal porcine reproductive and respiratory syndrome virus detection primers ORF6-F and ORF6-R:

[0062] ①RNA template preparation: Porcine reproductive and respiratory syndrome virus (PRRSV) positive samples were extracted. Positive samples included commercial live vaccines (Sinde Porcine Reproductive and Respiratory Syndrome Virus Live Vaccine, strain GD-R; Zhaofenghua Porcine Reproductive and Respiratory Syndrome Virus Live Vaccine, strain CH-1R), Porcine Reproductive and Respiratory Syndrome Virus ORF6 standard plasmid (synthesized by Sangon Biotech (Shanghai) Co., Ltd.), and samples confirmed to be positive for Porcine Reproductive and Respiratory Syndrome by sequencing results.

[0063] DNA / RNA nucleic acid extraction methods can be performed by referring to the instructions for the DNA / RNA nucleic acid extraction kit.

[0064] ②RT-PCR: Take out the one-step RNase system (purchased from Tiangen Biotech (Beijing) Technology Co., Ltd.), virus detection primers ORF6-F and ORF6-R, and ddH2O, place them in a 4℃ refrigerator to thaw completely, briefly and thoroughly shake, then centrifuge briefly, and add a tube of reverse transcription premixed mix to prepare the PCR-1 reaction solution. The volumes of each component of the PCR-1 reaction solution are shown in Table 1.

[0065] Table 1

[0066] name One-step reverse transcriptase One-tube reverse transcription premixed mix upstream primer Downstream primer <![CDATA[ddH2O]]> volume 1 ul 12.5 ul 1.25 ul 1.25 ul 6 ul

[0067] The total volume of the RT-PCR reaction system was 25 μL, including 22 μL of PCR-1 reaction solution and 3 μL of RNA template.

[0068] ③ Gel electrophoresis.

[0069] Gel electrophoresis results as follows Figure 2 As shown, the results indicate that commercial vaccines (Sinde GD-R and Mega-Feng Hua CH-1R), NADC30, NADC34, JX, and the positive control can all amplify a 112bp fragment, which demonstrates that the upstream and downstream primers of this detection method have good specificity and sensitivity.

[0070] The universal porcine reproductive and respiratory syndrome virus (PRRSV) detection primers ORF6-F and ORF6-R of this invention were used to amplify and compare typical genotype I and genotype II PRSV strains. The results are as follows: Figure 3 As shown in the figure. The results indicate that the universal porcine reproductive and respiratory syndrome virus (PRRSV) detection primers ORF6-F and ORF6-R of the present invention match both typical genotype I and genotype II PRRSV strains, enabling accurate detection of both PRRSV genotypes.

[0071] Example 3

[0072] Primer specificity test:

[0073] Positive samples included: NADC30, NADC34 and JX, commercial live vaccine strains (GD-R and CH-1R), nucleic acids from other viral disease positive samples (such as classical swine fever CSFV, pseudorabies PRV, porcine circovirus type 2 HCV2, porcine circovirus type 3 HCV3, epidemic diarrhea virus PEDV, transmissible gastroenteritis virus TGEV, rotavirus RV), and bacterial disease positive samples (such as infectious pleuropneumonia APP, streptococcus SS).

[0074] The above positive samples were subjected to real-time RT-PCR detection using the method described in Example 1. The detection results are as follows: Figure 4 As shown. Specificity test results showed that only samples with positive sequencing results for porcine reproductive and respiratory syndrome virus (PRRSV) such as NADC30, NADC34, JX, and commercial live vaccines (GD-R and CH-1R) could amplify characteristic curves. Other samples did not show amplification curves, indicating that the universal PRRSV detection primers ORF6-F and ORF6-R of this invention have excellent specificity.

[0075] Example 4

[0076] Establishment of the standard curve for the real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus:

[0077] The stock solution concentration of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 standard plasmid was 0.4 ng / µl. Following a 10-fold serial dilution, 4 × 10⁻⁶ plasmids were obtained. -2 4×10 -3 4×10 -4 4×10 -5 4×10 -6 4×10 -7 4×10 -8 ng / ul standard solution. These standard solutions were subjected to quantitative real-time RT-PCR according to the method in Example 1 to obtain amplification curves of porcine reproductive and respiratory syndrome virus, as shown below. Figure 5 As shown. With 4×10 -2 The concentration is ng / ul. Using the copy number calculation formula based on the plasmid molecular weight, the copy number is calculated to be 1.31 × 10⁻⁶. 7 Based on this data, a standard curve was plotted with the equation CT = -3.543LogCopies + 40.45, and a correlation coefficient R² = 0.9987. The standard curve is shown below. Figure 6 As shown.

[0078] Example 5

[0079] Sensitivity test of porcine reproductive and respiratory syndrome virus real-time fluorescent RT-PCR detection kit:

[0080] The standard plasmid of porcine reproductive and respiratory syndrome virus (PRRSV) ORF6 was serially diluted to prepare 4×10⁻⁶ plasmids. -2 4×10 -3 4×10 -4 4×10 -5 4×10 -6 4×10 -7 4×10 -8 4×10 -9 A standard solution of ng / µl was used for real-time RT-PCR reaction according to the method in Example 1. The results are as follows: Figure 7 As shown, 4×10 -9 The CT value of ng / µl was 37.09, which falls within the suspicious range of 35-40. Therefore, the fluorescence quantitative RT-PCR method established in this study exhibits high sensitivity.

[0081] Example 6

[0082] Sample validation experiment for the porcine reproductive and respiratory syndrome virus real-time fluorescent RT-PCR detection kit:

[0083] Samples exhibiting clinical symptoms of porcine reproductive and respiratory syndrome virus (PRRSV) were collected from pig farms, including porcine serum, tonsils, and lung tissue. The tests were performed according to the method described in Example 1, and the results are as follows: Figure 8 As shown.

[0084] Experimental results show that the detection kit of this invention can stably detect porcine reproductive and respiratory syndrome virus (PRRSV) samples. (Note: For samples that have just developed symptoms of PRS, the amount of RNA template needs to be increased. Usually, adding 1-2 μL to the normal amount of RNA template is sufficient for stable detection. For samples with CT values ​​between 37 and 40, it is recommended to re-extract nucleic acid and set up 3 parallel samples when adding the test samples. If two or more wells show CT values, it is considered positive; if only one well is positive, it is recommended that the pig farm resubmit the sample to determine whether there are problems such as sample contamination.)

[0085] The detection volume of porcine reproductive and respiratory syndrome virus from 2023 to August 2025 is as follows: Figure 9 As shown.

[0086] The number of samples tested for porcine reproductive and respiratory syndrome virus in Heilongjiang Province from 2024 to August 2025 is as follows: Figure 10 As shown.

[0087] The "Porcine Reproductive and Respiratory Syndrome Type 1-2 Multiplex Real-Time Quantitative PCR Detection Kit" commercially available from IDEXX was used for retesting. The positive detection accuracy of the real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus of this invention reached 98.7%. The unit price of the general kit of this invention is 245 / 50t, and the detection time is about 70 min. The detection cost and detection time are reduced by about 91.1% and 22.2% respectively compared with the commercially available product from IDEXX (IDEXX unit price is 5500 / 100t, detection time is about 90 min).

Claims

1. Universal primers and probes for detecting porcine reproductive and respiratory syndrome virus, characterized in that, The universal primers for detecting porcine reproductive and respiratory syndrome virus are ORF6-F and ORF6-R, and the detection probe is ORF6-P. The ORF6-F sequence is: 5'-TGCCGKTTGTGCTTGCTAG-3'; The ORF6-R sequence is: 5'-GGACGACAAATGCGTGGTTATC-3'; The ORF6-P sequence is: 5'-CGCAAGTACATTCTGG-3'; the 5' end of the detection probe ORF6-P sequence carries a fluorescent reporter group, and the 3' end carries a fluorescent quencher group.

2. The universal primers and probes for detecting porcine reproductive and respiratory syndrome virus according to claim 1, characterized in that, The detection probe sequence has a 5' fluorescent reporter group (FAM) and a 3' fluorescent quencher group (MGB).

3. A real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus, characterized in that, The real-time fluorescent RT-PCR detection kit includes universal porcine reproductive and respiratory syndrome virus detection primers ORF6-F and ORF6-R, and detection probe ORF6-P.

4. The real-time fluorescent RT-PCR detection kit for porcine reproductive and respiratory syndrome virus according to claim 3, characterized in that, The kit also includes a one-step RNase system, which consists of 5×one step u+ mix and one step u+ enzyme mix.

5. The use of the porcine reproductive and respiratory syndrome virus real-time fluorescent RT-PCR detection kit according to claim 3 or 4 in the preparation of products for diagnosing porcine reproductive and respiratory syndrome virus.

6. The use of the universal porcine reproductive and respiratory syndrome virus (PRRSV) detection primers described in claim 1 in the preparation of a real-time fluorescent RT-PCR detection kit for PRSV.

7. The application of the universal porcine reproductive and respiratory syndrome virus (PRRSV) detection primers and probes as described in claim 1 in the preparation of a real-time fluorescent RT-PCR detection kit for PRSV.

8. A method for identifying porcine reproductive and respiratory syndrome virus (PRRSV) for non-disease diagnostic purposes, characterized in that, Includes the following steps: Step 1: Preparation of the real-time RT-PCR reaction system and pre-reaction procedures; Step 2: Nucleic acid extraction from the test sample; Step 3: Quantitative Real-Time RT-PCR procedure; Step 4: The real-time PCR instrument reads the CT value of the test sample. If the CT value is <35, the test sample is considered positive. If a sample has a CT value between 35 and 40, it is considered suspicious and the nucleic acid needs to be extracted again for a second quantitative RT-PCR test. If the CT value of the second amplification is still between 35 and 40, it is considered positive. The virus detection primers in the real-time RT-PCR reaction system are ORF6-F and ORF6-R, and the detection probe is ORF6-P.

9. The method for identifying porcine reproductive and respiratory syndrome virus for non-disease diagnostic purposes according to claim 8, characterized in that, The reaction procedure for step three, quantitative real-time RT-PCR, is as follows: first stage reverse transcription: 55℃ for 15 min; second stage pre-denaturation: 95℃ for 30 s; third stage cycling: 95℃ for 10 s, 60℃ for 30 s, for a total of 45 cycles; fluorescence signal is collected at 60℃, and the FAM fluorescence channel is selected.

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