Sheep disease virus multiplex PCR detection primer, method and application

Abstract

The application belongs to the technical field of virus detection, and discloses a sheep disease virus multiplex PCR detection primer, method and application. The base sequence of the primer FMDV-F of the sheep foot-and-mouth disease virus is SEQ ID NO:1, the base sequence of the primer FMDV-R is SEQ ID NO:2; the base sequence of the primer ORFV-F of the sheep orf virus is SEQ ID NO:3, the base sequence of the primer ORFV-R is SEQ ID NO:4; the base sequence of the primer GTPV-F of the goat pox virus is SEQ ID NO:5, and the base sequence of the primer GTPV-R is SEQ ID NO:6. The application optimizes the multiplex PCR, determines the optimal reaction system and the optimal reaction procedure, and can accurately detect single or mixed infection of the sheep foot-and-mouth disease virus, the sheep orf virus and the goat pox virus, and has an important role in epidemiological investigation and epidemic disease prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of virus detection technology, and in particular relates to a primer, method and application for multiplex PCR detection of sheep disease virus. Background Technology

[0002] With the development of the livestock industry and the continuous expansion of breeding scale, the pressure of disease prevention and control is also increasing, especially for viral diseases in sheep, which pose a serious threat to the development of sheep farming due to their rapid spread and high pathogenicity. Foot-and-mouth disease (FMD), sheep pox, and goat pox are diseases caused by FMD virus, pox virus, and goat pox virus, respectively. Introduced breeds, immunization failure, and new variant virus strains are common factors contributing to infection with these viruses. Because these three diseases share similar clinical characteristics, and mixed infections are common, clinical diagnosis is difficult. Therefore, establishing rapid differential diagnostic methods is of great significance for the prevention and control of these three sheep diseases.

[0003] Foot-and-mouth disease (FMD), contagious ecthyma (CE), and goatpox (GTP) share similar clinical symptoms. FMD is an acute, febrile, highly contagious disease caused by the foot-and-mouth disease virus (FMDV), commonly known as "mouth sores." Domestic and wild cloven-hoofed animals are susceptible to this virus. Clinically, it manifests as vesicles on hairless areas such as the lips, tongue, inner surface, gums, cheek mucosa, lower extremities, and udder. These vesicles rupture, forming red ulcers. In severe cases, the hoof shell may detach, leading to lameness. Contagious ecthyma is a contagious, epitheliotropic disease caused by the orfvirus (ORFV), transmitted through direct contact. Goats, sheep, deer, camels, and other ruminants are susceptible to this disease. Clinical manifestations include pustules, vesicles, and crusts on the skin around the mouth, tongue, lips, udder, and muzzle. Goatpox is an acute, febrile, and contagious disease of sheep caused by goatpox virus (GTPV). Clinical manifestations include fever, conjunctivitis, rhinitis, excessive salivation, and vesicular and papular lesions on the skin and mucous membranes. With the rapid development of sheep farming, the types of sheep diseases have increased and become more difficult to control. Furthermore, mixed infections of multiple viruses exist; for example, Yan Xinmin et al. reported cases in my country of sheep co-infected with GTPV, ORFV, and contagious caprine pleuropneumonia.

[0004] Currently, there are two main types of detection methods for the aforementioned viruses. Serological methods include indirect enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, immunoenzyme agar diffusion assay, virus neutralization assay, and agar diffusion assay. Molecular biological methods include PCR technology and nucleic acid probe technology. Because these methods involve complex experimental procedures, high costs, and long processing times, establishing a rapid, efficient, and low-cost detection method is crucial for quickly identifying the type of virus infection in production practice and taking timely countermeasures. Multiplex PCR (Multiplex polymerase chain reaction, MPCR) refers to the simultaneous amplification of two or more target genes in a single reaction, and it has been widely studied due to its low cost, high efficiency, and high throughput. This technology is currently widely used in disease diagnosis and scientific research. However, there are currently no reports of using multiplex PCR to simultaneously detect three viruses: sheep foot-and-mouth disease virus, sheep pox virus, and goat pox virus.

[0005] Based on the above analysis, the problems and shortcomings of the existing technology are as follows: there are no reports on the existing technology of systematic multiplex PCR detection methods for FMDV, ORFV and GTPV pathogens at the same time. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a primer, method, and application for multiplex PCR detection of sheep disease viruses, particularly a primer, method, and application for multiplex PCR detection of sheep foot-and-mouth disease virus, sheep stomatitis virus, and goat pox virus.

[0007] This invention is achieved as follows: a multiplex PCR detection primer pair for sheep disease viruses. The primer FMDV-F for sheep foot-and-mouth disease virus has the base sequence SEQ ID NO: 1, and the primer FMDV-R has the base sequence SEQ ID NO: 2; the primer ORFV-F for sheep pox virus has the base sequence SEQ ID NO: 3, and the primer ORFV-R has the base sequence SEQ ID NO: 4; the primer GTPV-F for goat pox virus has the base sequence SEQ ID NO: 5, and the primer GTPV-R has the base sequence SEQ ID NO: 6.

[0008] Another object of the present invention is to provide a method for detecting sheep disease virus using multiplex PCR primers, the method comprising the following steps:

[0009] Step 1: Nucleic acid extraction and cDNA reverse transcription synthesis;

[0010] Step two: Optimize the conditions for multiplex PCR reactions;

[0011] Step 3: Perform multiplex PCR specificity and sensitivity tests;

[0012] Step four: Perform clinical sample testing using optimal PCR reaction conditions.

[0013] Furthermore, in step one, viral RNA or DNA is extracted from the tissue sample to be tested, the RNA is reverse transcribed into cDNA, and the viral DNA and cDNA are amplified using multiplex PCR detection primers.

[0014] Collect scabs, vesicle skin, vesicle fluid, or papule tissue from diseased sheep. Mix the tissue with physiological saline at a ratio of 1:2 to homogenize, and extract total nucleic acid using a centrifuged column-type viral DNA / RNA extraction kit.

[0015] Furthermore, in step one, the nucleic acid extraction of both the viral sample and the clinical sample to be tested was performed using a DNA / RNA extraction kit. The DNA sample was stored at -20°C. The RNA sample was reverse transcribed, and the obtained cDNA product was stored at -20°C, while the remaining RNA was stored at -80°C.

[0016] Furthermore, in step one, a 40 μL reverse transcription reaction system was used to reverse transcribe RNA using the HiScript cDNA first-strand synthesis kit; wherein, 4 μL of RNA template, 2 μL of random primers, 20 μL of 2×RT Mix, 4 μL of HiScript II Enzyme Mix, and RNase-free ddH2O were added to a total volume of 40 μL; the reverse transcription reaction program was: 25℃ for 5 min, 50℃ for 15 min, and 85℃ for 2 min.

[0017] Furthermore, in step two, the multiplex PCR reaction is performed in a 25 μL system; including 12.5 μL of 2×AceTaqMaster Mix; 0.5 μL each of FMDV-F and FMDV-R, with a concentration of 10 μmol / L; 1 μL each of ORFV-F and ORFV-R, with a concentration of 10 μmol / L; 0.5 μL each of GTPV-F and GTPV-R, with a concentration of 10 μmol / L; 1.5 μL of DNA / cDNA template; and sterile ddH2O to bring the total volume to 25 μL; the solute concentration in the 2×AceTaq Master Mix is ​​10 μmol / L.

[0018] Furthermore, in step two, the conditions for the multiplex PCR amplification reaction are as follows: the total volume of the reaction system is 25 μL; 1.5 μL each of the extracted tissue viral DNA or cDNA is added to the multiplex PCR reaction system, and sterile ddH2O is used to make up to a total volume of 25 μL; the multiplex PCR amplification reaction program is as follows: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, for a total of 30 cycles; 72℃ for 8 min.

[0019] Furthermore, in step three, the established optimal conditions for multiplex PCR reaction were used, with cDNA or DNA from samples of FMDV, ORFV, GTPV, bluetongue virus, peste des petits ruminants virus, Clostridium perfringens, Mycobacterium tuberculosis of sheep, and Mycoplasma caprineis as templates, and sterile water as a negative control, to detect the specificity of multiplex PCR.

[0020] Under the determined optimal PCR reaction conditions, the DNA or cDNA of the three viruses were measured and diluted 10-fold, and the PCR sensitivity was tested using the diluted DNA or cDNA of the three viruses as templates.

[0021] Another object of the present invention is to provide the application of the aforementioned sheep disease virus multiplex PCR detection primers in the preparation of single or mixed infection detection kits for sheep foot-and-mouth disease virus, sheep stomatitis virus and goatpox virus.

[0022] Furthermore, by performing agarose gel electrophoresis on the multiplex PCR products, the presence of a target band of the expected size on the electrophoresis pattern was observed to determine whether the tested sample was a single or mixed infection; among them, the expected band size for sheep foot-and-mouth disease virus was 544 bp, the expected band size for sheep pox virus was 361 bp, and the expected band size for goatpox virus was 222 bp.

[0023] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0024] First, addressing the technical problems existing in the prior art and the difficulty of solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0025] This invention provides specific primers, methods, and applications for multiplex PCR detection of foot-and-mouth disease virus (FMDV), sheep poxvirus, and goatpox virus (WPV), enabling the simultaneous detection of these three pathogens. By optimizing multiplex PCR, this invention determines the optimal reaction system (primer and template ratios) and the optimal reaction procedure (annealing temperature). This invention can accurately detect single or mixed infections of FMDV, WPV, and WPV, playing a crucial role in epidemiological investigations and disease control.

[0026] Second, considering the technical solution as a whole or from a product perspective, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows:

[0027] This invention, through multiple experimental verifications, establishes for the first time a multiplex PCR method capable of simultaneously detecting FMDV, ORFV, and GTPV. It mainly involves primer pairs for multiplex PCR detection of sheep foot-and-mouth disease virus, sheep pox virus, and goat pox virus, as well as detection methods using primer pairs and applications of primers. This invention provides technical support for rapid diagnosis and epidemiological investigation of these three viruses.

[0028] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0029] The technical solution of this invention fills a technological gap in the industry both domestically and internationally:

[0030] Qin Min et al. established multiplex PCR detection methods for bluetongue disease, FMDV, peste des petits ruminants (PPR), and vesicular stomatitis. He Yapeng et al. established multiplex RT-PCR methods involving FMDV, bluetongue disease, and PPR, both of which have shown good results in clinical applications. Because sheep pox is prevalent in Northwest China, He Yapeng et al. established multiplex PCR detection methods for sheep pox virus, GTPV, and ORFV. Although previous studies on multiplex PCR have all involved the analytes of this invention, FMDV, ORFV, and GTPV, there are currently no reports on systematic multiplex PCR detection methods for FMDV, ORFV, and GTPV pathogens simultaneously. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a flowchart of the multiplex PCR detection method for sheep disease viruses provided in this embodiment of the invention;

[0033] Figure 2 This is an electrophoresis image of a single or mixed sample of sheep foot-and-mouth disease virus, sheep pox virus, and goatpox virus detected by multiplex PCR according to an embodiment of the present invention; wherein, M is a standard with a molecular weight of 2000; the 544bp band represents sheep foot-and-mouth disease virus; the 361bp band represents sheep pox virus; and the 222bp band represents goatpox virus.

[0034] Figure 3 This is an electrophoresis image of a multiplex PCR specific assay provided in this embodiment of the invention; wherein, M is a standard with a molecular weight of 2000; 1 to 4 are single or mixed samples of sheep foot-and-mouth disease virus, sheep orthole virus, and goatpox virus; 5 is a bluetongue virus sample; 6 is a small ruminant plague virus sample; 7 is a Clostridium perfringens sample; 8 is a sheep tuberculosis sample; 9 is a caprine mycoplasma sample; 10 is a negative control;

[0035] Figure 4 This is an electrophoresis image of the multiplex PCR sensitivity assay provided in this embodiment of the invention; wherein, M is a standard with a molecular weight of 2000; 1 to 8 are extracted sheep foot-and-mouth disease virus, sheep orthopnea virus and goatpox virus nucleic acid templates that are serially diluted 10-fold from 10 8 Up to 10 1 Copy / μL; Sheep foot-and-mouth disease virus nucleic acid from 5.47 × 10⁻⁶ 8 10-fold serial dilution to 5.47 × 10⁻⁶ copies / μL 1 Copy / μL; sheep pox virus nucleic acid from 1.36 × 10⁻⁶ 8 10-fold serial dilution to 1.36 × 10⁻⁶ copies / μL 1 Copies / μL; Goatpox virus nucleic acid consists of 3.19 × 10⁻⁶ copies / μL. 8 10-fold serial dilution to 3.19 × 10⁻⁶ copies / μL 1 Copy / μL. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] To address the problems existing in the prior art, this invention provides primers, methods, and applications for multiplex PCR detection of sheep disease viruses. The invention will be described in detail below with reference to the accompanying drawings.

[0038] To enable those skilled in the art to fully understand how the present invention is specifically implemented, this section provides an explanatory description of the embodiments that expand upon the technical solutions of the claims.

[0039] like Figure 1As shown, the multiplex PCR detection method for sheep disease viruses provided in this embodiment of the invention includes the following steps:

[0040] S101, primers were designed and nucleic acid extraction and cDNA reverse transcription synthesis were performed;

[0041] S102, to optimize the conditions for multiplex PCR reactions;

[0042] S103 was used for multiplex PCR specificity and sensitivity tests.

[0043] S104 uses optimal PCR reaction conditions for clinical sample testing.

[0044] The base sequence of the sheep foot-and-mouth disease virus primer FMDV-F provided in this embodiment of the invention is SEQ ID NO: 1, and the base sequence of FMDV-R is SEQ ID NO: 2; the base sequence of the sheep orthopnea virus primer ORFV-F is SEQ ID NO: 3, and the base sequence of ORFV-R is SEQ ID NO: 4; the base sequence of the goat pox virus primer GTPV-F is SEQ ID NO: 5, and the base sequence of GTPV-R is SEQ ID NO: 6.

[0045] The primer sequence for foot-and-mouth disease virus (FMDV) in the multiplex PCR detection primer pairs for sheep foot-and-mouth disease virus, sheep pox virus, and goat pox virus provided in this embodiment of the invention is as follows:

[0046] FMDV-F: 5'-GTGACGCCATGAACATC-3'

[0047] FMDV-R: 5'-GCGAGTCCTGCCACGGA-3'

[0048] Primer sequence for Oral Herpes Virus (ORFV):

[0049] ORFV-F: 5'-AGTCCGCGAAGAAGTTTTTGT-3'

[0050] ORFV-R: 5'-TTCATGAGGTCCCAGGCCAG-3'

[0051] Primer sequence for goat poxvirus (GTPV):

[0052] GTPV-F: 5'-TATTTTGTGCGGAAACGT-3'

[0053] GTPV-R: 5'-ATTATAGGAATTGTTGAG-3'

[0054] In this embodiment of the invention, total DNA and total RNA are extracted from the clinical samples to be tested. The total RNA is reverse transcribed into cDNA, and the DNA or cDNA of the sample is amplified using the reaction system and reaction program of the multiplex PCR primer pair.

[0055] The multiplex PCR reaction system provided in this embodiment of the invention is as follows: 12.5 μL of 2×AceTaq MasterMix; 1 μL of primers for sheep foot-and-mouth disease virus, including 0.5 μL each of FMDV-F and FMDV-R (concentration of 10 μmol / L); 2 μL of primers for sheep pox virus, including 1 μL each of ORFV-F and ORFV-R (concentration of 10 μmol / L); 1 μL of primers for sheep pox virus, including 0.5 μL each of GTPV-F and GTPV-R (concentration of 10 μmol / L); 1.5 μL of DNA / cDNA template; sterile ddH2O to a total volume of 25 μL; the solute concentration in the 2×AceTaq Master Mix is ​​10 μmol / L; the amplification reaction program is: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, for a total of 30 cycles; 72℃ for 8 min.

[0056] The extraction method provided in this embodiment of the invention involves collecting scabs, vesicles, vesicle fluid, or papule tissue from diseased sheep, mixing the tissue with physiological saline at a ratio of 1:2 to homogenize, and extracting total nucleic acid using a centrifuged column-type viral DNA / RNA extraction kit.

[0057] The reverse transcription conditions provided in this embodiment of the invention are as follows: The RNA is reverse transcribed using the HiScript cDNA first-strand synthesis kit; a 40 μL system is used, and the reaction mixture consists of: 4 μL RNA template, 2 μL random primers, 20 μL 2×RT Mix, 4 μL HiScript II Enzyme Mix, and RNase-free ddH2O to a total volume of 40 μL. The reaction program is: 25℃ for 5 min, 50℃ for 15 min, and 85℃ for 2 min.

[0058] To demonstrate the inventiveness and technical value of the technical solution of this invention, this section provides specific product or related technology application examples of the technical solution claimed.

[0059] The application of this invention provides a multiplex PCR detection primer pair for sheep foot-and-mouth disease virus, sheep thrush virus, and goat pox virus in the preparation of a kit for detecting single or mixed infections of sheep foot-and-mouth disease virus, sheep thrush virus, and goat pox virus.

[0060] In this embodiment of the invention, multiplex PCR products are subjected to agarose gel electrophoresis. The presence of a target band of the expected size on the electrophoresis pattern is used to determine whether the sample is a single or mixed infection. The expected band size for sheep foot-and-mouth disease virus is 544 bp, the expected band size for sheep pox virus is 361 bp, and the expected band size for goatpox virus is 222 bp.

[0061] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes them in conjunction with the data, charts and other information of the experimental process.

[0062] 1. Materials and Methods

[0063] 1.1 Test Materials

[0064] The ORFV, GTPV, bluetongue virus, small ruminant virus, Clostridium perfringens, Mycobacterium tuberculosis of sheep, and Mycoplasma caprineis samples involved in the embodiments of this invention were all identified and preserved by our laboratory. The FMDV vaccine strain was purchased from Inner Mongolia Jinyu Baoling Biological Pharmaceutical Co., Ltd. A total of 132 anticoagulated blood samples and 8 identified positive scab tissue samples from sheep ulcers were collected from various large-scale sheep farms in Guizhou Province from December 2021 to November 2022.

[0065] 1.2 Main Reagents and Instruments

[0066] Nucleic Acid Extraction Kit (DNA / RNA Extraction Kit), Gel Extraction Kit Gel DNA Extraction Mini Kit, Plasmid Extraction Kit, RapidLyse Plasmid Mini Kit, Reverse Transcription Kit II. The 1st Strand cDNA Synthesis Kit, DNA polymerase mixture 2×AceTaq MasterMix (Dye Plus), gene cloning vector ClonExpress MultiS One Step Cloning Kit, and DH5α chemicompetent cells were all Novizan products; the DL2000 DNA Marker was a product of Tiangen Company; the PCR instrument was a product of BIO-RAD; and the gel electrophoresis instrument was a product of Beijing Liuyi Company.

[0067] 1.3 Primer Design

[0068] Download the complete genome sequences of different serotypes of FMDV, ORFV, and GTPV that have been uploaded to NCBI. Use the clustlW software to perform multiple sequence alignment of the genome sequences of different serotypes of the same virus. Design primers for highly conserved regions. The primer sequences are shown below:

[0069] Primer sequence for sheep foot-and-mouth disease virus (FMDV):

[0070] FMDV-F: 5'-GTGACGCCATGAACATC-3'

[0071] FMDV-R: 5'-GCGAGTCCTGCCACGGA-3'

[0072] Primer base sequence for Oral Flood Virus (ORFV):

[0073] ORFV-F: 5'-AGTCCGCGAAGAAGTTTTTGT-3'

[0074] ORFV-R: 5'-TTCATGAGGTCCCAGGCCAG-3'

[0075] Primer base sequences for goat pox virus (GTPV):

[0076] GTPV-F: 5'-TATTTTGTGCGGAAACGT-3'

[0077] GTPV-R: 5'-ATTATAGGAATTGTTGAG-3'

[0078] 1.4 Nucleic acid extraction and cDNA synthesis

[0079] DNA / RNA from three viruses were extracted using a DNA / RNA Extraction Kit. The II 1st Strand cDNA Synthesis Kit is used for reverse transcription of RNA: 4 μL RNA template, 2 μL random primers, 20 μL 2×RT Mix, 4 μL HiScript II Enzyme Mix, and RNase-free ddH2O to a total volume of 40 μL. The reverse transcription program is: 25℃ for 5 min, 50℃ for 15 min, and 85℃ for 2 min. The obtained cDNA or total DNA product is stored at -20℃, and the remaining RNA is stored at -80℃.

[0080] 1.5 Optimization of Multiplex PCR Reaction Conditions

[0081] The reaction system was optimized. Multiplex PCR was performed in a total volume of 25 μL. The amount of primers added for each virus detection pair was adjusted to determine the optimal primer concentration. The reaction conditions were as follows: 12.5 μL of 2×AceTaq MasterMix; 1 μL of primers for sheep foot-and-mouth disease virus, including 0.5 μL each of FMDV-F and FMDV-R (concentration of 10 μmol / L); 2 μL of primers for sheep pox virus, including 1 μL each of ORFV-F and ORFV-R (concentration of 10 μmol / L); 1 μL of primers for sheep pox virus, including 0.5 μL each of GTPV-F and GTPV-R (concentration of 10 μmol / L); 1.5 μL each of FMDV cDNA, ORFV DNA, and GTPV DNA templates; and sterile ddH2O to a total volume of 25 μL. The amplification reaction program was as follows: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, for a total of 30 cycles; 72℃ for 8 min.

[0082] 1.6 Multiplex PCR Specificity Assay

[0083] Using the established optimal conditions for multiplex PCR, cDNA or DNA samples of FMDV, ORFV, GTPV, bluetongue virus, peste des petits ruminants virus, Clostridium perfringens, Mycobacterium tuberculosis of sheep, and Mycoplasma caprineis were used as templates, with sterile water as a negative control to detect the specificity of multiplex PCR.

[0084] 1.7 Multiplex PCR Sensitivity Test

[0085] Under the determined optimal PCR reaction conditions, the DNA or cDNA of the three viruses were measured and diluted 10-fold, and used as templates to detect PCR sensitivity.

[0086] 1.8 Detection of clinical samples

[0087] Optimal PCR reaction conditions were used to detect whole blood samples, pathological tissue samples, and whole blood simulants collected from different sheep farms in Guizhou Province. This included 132 anticoagulated blood samples, 8 positive sheep scab samples, and three ORFV simulants and one GTPV simulant sample prepared by mixing 100 μL of virus cell culture supernatant with 200 μL of negative whole blood. One FMDV simulant sample was prepared by mixing a 10-fold diluted FMDV vaccine strain with 200 μL of negative whole blood. Two mixed simulant samples were prepared by mixing equal volumes of the three virus solutions with 100 μL and 200 μL of negative whole blood. Nucleic acids were extracted from the samples and detected using both the established multiplex PCR method and conventional PCR.

[0088] 2. Results

[0089] 2.1 Establishment of optimal multiplex PCR reaction conditions

[0090] The optimal multiplex PCR reaction system, as verified by experiments, is as follows: total volume 25 μL; 12.5 μL 2×AceTaqMaster Mix; 0.5 μL each of FMDV-F and FMDV-R; 1 μL each of ORFV-F and ORFV-R; 0.5 μL each of GTPV-F and GTPV-R; 1.5 μL each of FMDV cDNA, ORFV DNA, and GTPV DNA template; sterile ddH2O to bring the total volume to 25 μL. The optimal amplification reaction program is: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, for a total of 30 cycles; 72℃ for 8 min. The results showed that the target band sizes of FMDV, ORFV, and GTPV were consistent with expectations, at 544 bp, 361 bp, and 222 bp, respectively. Figure 2 As shown.

[0091] 2.2 Specificity test

[0092] The detection of FMDV, ORFV, and GTPV nucleic acid samples all yielded target bands of the expected size. Amplification of other sheep pathogen nucleic acids under the same conditions did not reveal specific bands. This result indicates that the established multiplex PCR method has good specificity. (See results below.) Figure 3 As shown.

[0093] 2.3 Sensitivity Test

[0094] Viral nucleic acid samples were serially diluted 10-fold and used as templates for amplification under established multiplex PCR conditions. The results showed that the detection limits of this multiplex PCR for FMDV, ORFV, and GTPV were 5.47 × 10⁻⁶. 3 Copies / μL, 1.36 × 10 4 Copies / μL, 3.19 × 10 3 Copy / μL, such as Figure 4 As shown.

[0095] 2.4 Clinical Sample Testing

[0096] Optimal multiplex PCR conditions were used to detect ORFV infection in 132 whole blood samples, 7 whole blood simulant samples, and 8 positive sheep stomatitis scab tissue samples collected from different sheep farms in Guizhou Province. The results are shown in Table 1. Multiplex PCR detected one case of ORFV infection in the whole blood sample, 3 simulant samples of FMDV, 5 samples of ORFV, 3 samples of GTPV, and 12 mixed samples of FMDV, ORFV, and GTPV. Eight tissue samples were found to be infected with ORFV. The concordance rate with conventional PCR results was 100%.

[0097] Table 1. Detection results of clinical samples

[0098]

[0099] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A primer for detecting capripoxvirus by multiplex PCR, characterized by, In the primer pair for detecting multiple PCR of sheep disease virus, the base sequence of the primer FMDV-F of foot-and-mouth disease virus is SEQ ID NO: 1, and the base sequence of the primer FMDV-R is SEQ ID NO: 2; the base sequence of the primer ORFV-F of orf virus is SEQ ID NO: 3, and the base sequence of the primer ORFV-R is SEQ ID NO: 4; the base sequence of the primer GTPV-F of goat pox virus is SEQ ID NO: 5, and the base sequence of the primer GTPV-R is SEQ ID NO: 6; The primer is used for a 25 μL system of multiple PCR reaction; wherein, 2×AceTaq Master Mix is 12.5 μL; FMDV-F and FMDV-R are each 0.5 μL, and the concentration is 10 μmol / L; ORFV-F and ORFV-R are each 1 μL, and the concentration is 10 μmol / L; GTPV-F and GTPV-R are each 0.5 μL, and the concentration is 10 μmol / L; DNA / cDNA template is 1.5 μL; sterilized ddH2O is supplemented to a total volume of 25 μL; the concentration of solutes in 2×AceTaq Master Mix is 10 μmol / L; the multiple PCR amplification reaction program is: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, a total of 30 cycles; 72℃ for 8 min.

2. The application of the multiple PCR detection primer of sheep disease virus in claim 1 in the preparation of a single or mixed infection detection kit for foot-and-mouth disease virus, orf virus and goat pox virus.

3. The use of the primers for multiple PCR detection of sheep disease viruses according to claim 2 in the preparation of a single or mixed infection detection kit for foot-and-mouth disease virus, orf virus and capripox virus, characterized in that, By performing agarose gel electrophoresis on the multiple PCR product, whether the expected size of the target band is observed to determine whether the detected sample is single or mixed infection; In the primer pair for detecting multiple PCR of sheep disease virus, the base sequence of the primer FMDV-F of foot-and-mouth disease virus is SEQ ID NO: 1, and the base sequence of the primer FMDV-R is SEQ ID NO: 2; the base sequence of the primer ORFV-F of orf virus is SEQ ID NO: 3, and the base sequence of the primer ORFV-R is SEQ ID NO: 4; the base sequence of the primer GTPV-F of goat pox virus is SEQ ID NO: 5, and the base sequence of the primer GTPV-R is SEQ ID NO: 6; The primer is used for a 25 μL system of multiple PCR reaction; wherein, 2×AceTaq Master Mix is 12.5 μL; FMDV-F and FMDV-R are each 0.5 μL, and the concentration is 10 μmol / L; ORFV-F and ORFV-R are each 1 μL, and the concentration is 10 μmol / L; GTPV-F and GTPV-R are each 0.5 μL, and the concentration is 10 μmol / L; DNA / cDNA template is 1.5 μL; sterilized ddH2O is supplemented to a total volume of 25 μL; the concentration of solutes in 2×AceTaq Master Mix is 10 μmol / L; the multiple PCR amplification reaction program is: 95℃ for 5 min; 95℃ for 10 s, 57℃ for 30 s, 72℃ for 40 s, a total of 30 cycles; 72℃ for 8 min.

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