A MALDI-TOF mass spectrometry method for detecting bovine lumpy skin disease virus, goat pox virus and sheep pox virus

Abstract

The present invention relates to the field of pathogenic microorganism detection, and in particular to a MALDI-TOF mass spectrometry detection method for bovine nodular skin disease virus, goat pox virus and sheep pox virus, and a primer combination used in the method. The present invention combines multiplex PCR amplification technology, a dual-gene target strategy, a single base extension reaction, and the high-precision analysis capability of MALDI-TOF MS to provide an efficient method for simultaneously performing multi-target accurate detection of three sheep pox viruses. The present invention adopts 2 pairs of specific amplification primers to target and amplify target gene fragments, and is supplemented by 2 extension primers for subsequent single base extension reactions, and then accurately measures the molecular weight difference generated by a single base in the extension product on MALDI-TOF MS. It can be achieved in a single-tube reaction system that not only efficiently identifies whether there are three sheep pox viruses in the sample, but also can further subdivide and clearly report whether the specific infection is one, several or all of the three viruses, thereby greatly improving the comprehensiveness, specificity and accuracy of the detection.

Description

Technical Field

[0001] The present invention relates to the field of pathogenic microorganism detection, and in particular to a MALDI-TOF mass spectrometry detection method for bovine lumpy skin disease virus, goat pox virus and sheep pox virus, and a primer combination used in the method. Background Art

[0002] Lumpy skin disease virus (LSDV), goatpoxvirus (GTPV), and sheeppox virus (SPPV) are all members of the Capripoxvirus (CaPV) genus. Infection with these viruses can cause symptoms such as fever, skin or mucous membrane nodules (or papules), and lymphadenopathy. Poxvirus-induced diseases can significantly reduce production performance and, in severe cases, lead to death. For example, lumpy skin disease in cattle can cause deep pores in the skin, rendering the hide unusable, leading to infertility in males and miscarriage in females, a significant decrease in milk production in dairy cows, and, in severe cases, death. Goatpox and sheeppox viruses can have mortality rates as high as 100% in highly susceptible sheep breeds, and epidemics of these three diseases have caused significant economic losses worldwide. Due to the economic importance of cattle, sheep, and goat farming, and the potential for rapid cross-border spread of these viruses, lumpy skin disease in cattle, goatpox, and sheeppox are all notifiable diseases of the World Organization for Animal Health (OIE).

[0003] Currently, the differential diagnosis of bovine nodular dermatitis, goat pox, and sheep pox typically relies on classic virological isolation techniques and serological analysis strategies. The core of viral isolation technology involves collecting samples of suspected lesions, propagating the virus in a cell culture system, and then using electron microscopy to analyze the morphological and structural characteristics of the resulting viral particles. Although this strategy can accurately identify and subtype viruses, it suffers from significant drawbacks: it is time-consuming (often requiring days to weeks), has complex procedures, and requires rigorous expertise from laboratory personnel, thus limiting its widespread adoption and application in clinical practice and large-scale screening. Because LSDV, GTPV, and SPPV strains exhibit high antigenic similarity, traditional serological methods are unable to effectively distinguish between them. However, LSDV, GTPV, and SPPV strains differ in their genetic sequences. Therefore, PCR technology, with its advantages of ease of operation, rapid detection, high sensitivity, and strong specificity, has shown great potential for rapid screening of clinical samples. It not only significantly shortens the detection cycle but also reduces the requirements for sample quality, providing strong support for early diagnosis of the disease. However, PCR is generally limited to detecting a single or a few viruses, making it difficult to fully cover and accurately genotype viral lineages. Furthermore, while high-throughput sequencing (HTS) can accurately identify viruses, its high cost, complex workflow, and relatively long data processing times hinder its widespread application in rapid diagnostics.

[0004] Therefore, there is an urgent need to develop a low-cost detection method with high specificity, high sensitivity, high throughput and relatively simple operation to detect, identify and further type bovine lumpy dermatitis, goat pox and sheep pox viruses. Summary of the Invention

[0005] In view of this, the present invention combines multiplex PCR amplification technology, dual-gene target strategy, single-base extension reaction, and the high-precision analysis capability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to develop an efficient method that can simultaneously and accurately detect three sheep pox viruses with multiple targets. Specifically, the present invention designs and applies two pairs of specific amplification primers to target the amplification of target gene fragments, and supplements them with two extension primers for subsequent single-base extension reactions. By accurately measuring the molecular weight difference of a single base in the extension product on MALDI-TOF MS, it can be achieved in a single-tube reaction system. It can not only efficiently identify whether the sample is infected with the three sheep pox viruses (i.e., LSDV, GTPV, and SPPV), but also further subdivide and clearly report whether the specific infection is one, several, or all of these three viruses, thereby greatly improving the comprehensiveness, specificity, and accuracy of the detection.

[0006] The technical solution of the present invention is achieved as follows:

[0007] In a first aspect, the present invention provides a primer composition for identifying bovine lumpy skin disease virus, goat pox virus and sheep pox virus, the composition comprising amplification primers and single-base extension primers, the amplification primers comprising SEQ ID NOs: 1-4, and the single-base extension primers comprising SEQ ID NOs: 5-6.

[0008] The selection of dual gene targets and primer design ideas include the following:

[0009] The P32 protein is a major structural protein located on the surface of the capripoxvirus membrane. It is a protein unique to the capripoxvirus genus, and the gene encoding this protein is specific to the capripoxvirus genus. The GPCR (G protein-coupled chemokine receptor) gene is somewhat conserved in the capripoxvirus genus, but there are also sufficient sequence differences to distinguish different types of viruses. Based on the conserved sequence of the selected target gene, specific amplification primers are designed, and a 10-base universal sequence ACGTTGGATG is added to the 5' end of each amplification primer. A single-base extension primer is designed in the conserved sequence region in the amplification region, and a designed and determined base is allowed to be extended at the 3' end of the single-base extension primer as a specific sequence marker for different capripoxviruses. The GPCR gene extension base site selection is as follows: Figure 1 As shown, from the sequence comparison results of the representative strains of the three poxviruses, it can be seen that when the extension primer is designed before the mutated base, the extended base of the goatpox virus is T, the extended base of the sheeppox virus is A, and the extended base of the bovine lumpy skin disease virus is G.

[0010] In a second aspect, the present invention provides an application of the primer composition, wherein the application is non-disease treatment and / or diagnosis, and the primer composition is used for at least one of (A1)-(A2): (A1) preparing diagnostic products for bovine nodular dermatitis, goat pox and / or sheep pox; (A2) preparing detection products for bovine nodular dermatitis virus, goat pox virus and sheep pox virus.

[0011] In a third aspect, the present invention provides a kit comprising the primer composition.

[0012] Furthermore, the kit also includes PCR amplification reaction reagents. In some embodiments, the components of the PCR amplification reaction kit include: Mg 2+ , dNTPs, H2O, DNA polymerase and buffer or a combination of one or more.

[0013] The kit also includes a dephosphorylation reagent. In some embodiments, the dephosphorylation reagent includes alkaline phosphatase and a buffer. Furthermore, the alkaline phosphatase is shrimp alkaline phosphatase (SAP enzyme).

[0014] The kit also includes a single base extension reaction reagent. In some embodiments, the components of the single base extension reaction reagent include: dNTPs, DNA polymerase, H2O, buffer and Mg 2+ A combination of one or more of .

[0015] In a fourth aspect, the present invention provides a MALDI-TOF mass spectrometry method for detecting bovine lumpy skin disease virus, goat pox virus, and sheep pox virus. The detection method is for non-disease treatment and / or diagnosis, and uses the conserved region of the P32 gene and the variable region of the GPCR gene as dual targets for detection, comprising the following steps:

[0016] (S1) performing PCR amplification on the sample to be tested using SEQ ID NOs: 1-4 as amplification primers to obtain an amplified product;

[0017] (S2) dephosphorylating the amplified product using alkaline phosphatase to obtain a dephosphorylated product;

[0018] (S3) using SEQ ID NO: 5-6 as a single base extension primer, performing single base extension on the dephosphorylated product to obtain an extension product;

[0019] (S4) purifying the extension product;

[0020] (S5) MALDI-TOF mass spectrometry detection.

[0021] The sample to be tested is the extracted nucleic acid. Specifically, in the step (S4), the purification includes one of column chromatography purification, PAGE purification, magnetic bead purification and HPLC purification. The column chromatography purification includes resin desalting purification.

[0022] In some preferred embodiments, in step (S1), the PCR amplification reaction is a multiplex PCR amplification reaction, and the reaction procedure includes: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 30 s, 45 cycles; and storage at 4°C.

[0023] In some preferred embodiments, in the step (S3), the reaction procedure of single base extension includes (see Table 6): pre-denaturation at 95°C for 30s; denaturation at 95°C for 5s, (annealing at 52°C for 5s, extension at 80°C for 5s, 5 cycles), 40 cycles; extension at 72°C for 3min, and storage at 4°C.

[0024] In some preferred embodiments, in the step (S5), the molecular weight of the purified product obtained in the step (S4) is detected by MALDI-TOF mass spectrometry, and bovine lumpy skin disease virus, goat pox virus and sheep pox virus are identified based on the molecular weight difference.

[0025] In some preferred embodiments, the molecular weight information of each single-base extension primer and the single-base extension product is imported into the mass spectrometer operating software, and the result is determined based on whether the single-base extension primer is extended and a specific extension peak (i.e., a peak that matches the expected extension product molecular weight) and the base appear.

[0026] The mass spectrometry analysis results correspond to two peak positions: the single-base extension primer and the single-base extension product. If the primer extension conversion rate is 100%, the single-base extension primer peak disappears. If a single peak appears at the molecular weight position corresponding to any extension product of a target and the signal-to-noise ratio (SNR) is greater than 6, the target is judged to be positive. If there is no peak at the extension product position corresponding to the target or the signal-to-noise ratio of the peak is less than 6, the target is judged to be negative.

[0027] The sequences and molecular weight (Mass) of the amplification primers, extension primers, and products are shown in Table 1 below:

[0028] Table 1

[0029]

[0030] The shaded portion of the possible extension product sequence represents the extended base. In the interpretation rules, A / G / T represent different extended bases. LSDV, GTPV, and SPPV correspond to bovine lumpy skin disease virus, goat pox virus, and sheep pox virus, respectively.

[0031] The technical solution of the present invention has the following advantages and beneficial effects:

[0032] The present invention designs and utilizes two pairs of specific amplification primers to target and amplify target gene fragments, supplemented by two extension primers to support subsequent single-base extension reactions. By utilizing MALDI-TOF mass spectrometry to precisely measure molecular weight differences caused by single bases in the extension products, this method can efficiently identify the presence of three different viruses of the genus Capripoxvirus (specifically, bovine lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV)) in a sample in a single reaction system, and further specifically genotype the infected viruses, thereby significantly improving the comprehensiveness, specificity, and accuracy of the test.

[0033] This method demonstrates precise typing capabilities, with concise and efficient interpretation rules. By optimizing the selection of amplification sites and the addition of modified bases, this method ensures effective differentiation of extended probe molecular weights within the detection range, achieving the goal of multiplexed detection. The ability to support multiplexed detection per well far exceeds that of traditional fluorescent quantitative PCR methods. Furthermore, this method does not rely on fluorescent dyes, breaking away from the limitations of fluorescent channels and improving detection flexibility and accuracy.

[0034] To improve detection efficiency and throughput, the present invention is compatible with high-density 384-well chips, enabling single-well reactions to complete the entire experiment and data analysis in a relatively short period of time, making it suitable for rapid screening of large-scale samples. Furthermore, the flexible sample loading process and the multiple-use chip further reduce costs. Researchers can adjust the detection reagents according to actual needs, and the simple operation eliminates the need for complex professional guidance, enhancing the practicality and scalability of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 Schematic diagram of the GPCR gene sequence alignment of representative strains of bovine lumpy skin disease virus, goat pox virus, and sheep pox virus;

[0037] Figure 2 This is the MALDI-TOF mass spectrometry test result of bovine lumpy skin disease virus (LSDV) sample;

[0038] Figure 3 This is the MALDI-TOF mass spectrometry test result of goatpox virus (GTPV) sample;

[0039] Figure 4 This is the MALDI-TOF mass spectrometry test result of sheep pox virus (SPPV) sample;

[0040] Figure 5 The results of MALDI-TOF mass spectrometry detection of a mixed sample of bovine lumpy skin disease virus (LSDV) and goat pox virus (GTPV);

[0041] Figure 6 The results of MALDI-TOF mass spectrometry detection of a mixed sample of bovine lumpy skin disease virus (LSDV), goat pox virus (GTPV) and sheep pox virus (SPPV);

[0042] Figure 7This is the MALDI-TOF mass spectrometry test result of ovine infectious pustular virus (ORFV) sample;

[0043] Figure 8 This is the MALDI-TOF mass spectrometry result of goatpox virus 10 copies / mL;

[0044] Figure 9 This is the MALDI-TOF mass spectrometry detection result of bovine lumpy skin disease virus 10 copies / mL. DETAILED DESCRIPTION

[0045] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0047] The methods for obtaining the various biological materials described in the examples merely provide experimental methods for achieving the disclosed objectives and should not be construed as limiting the sources of the biological materials used in the present invention. In fact, the sources of biological materials used are diverse, and any legally and ethically accessible biological material may be substituted for and used as indicated in the examples.

[0048] The terms "including" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps that are not listed, or may optionally include other steps that are inherent to these processes, methods, products or devices. The "plurality" mentioned in the present invention refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the related objects before and after are in an "or" relationship.

[0049] Example 1 Application of the detection method, primer combination or kit provided by the present invention in identifying bovine lumpish skin disease virus, goat pox virus and sheep pox virus

[0050] 1. Dual target gene selection and design of specific amplification primers and single-base extension primers

[0051] First, the gene sequences of representative strains of bovine lumpy skin disease virus, goat pox virus and sheep pox virus were downloaded from the GenBank database after complete annotation as reference sequences, and the P32 and GPCR genes were selected as dual targets.

[0052] The P32 protein is a major structural protein located on the surface of the capripoxvirus membrane and is unique to the capripoxvirus genus. The gene encoding this protein is specific to the capripoxvirus genus. GPCR (G protein-coupled chemokine receptor) genes are somewhat conserved within the capripoxvirus genus, but there is also sufficient sequence diversity to distinguish between different virus species.

[0053] After sequence alignment and phylogenetic tree analysis, the conserved regions of the P32 gene were identified, and mutation sites of the GPCR gene were selected. Specific amplification primers were designed based on the conserved sequences of the selected target genes. A 10-base universal sequence of ACGTTGGATG (as shown in Table 1) was added to the 5' end of each amplification primer. A single-base extension primer was designed in the conserved sequence region of the amplification region. At the 3' end of the single-base extension primer, a designed and determined base mutation site was allowed to be extended as a specific sequence marker for different capripoxviruses. The base site extension of the GPCR gene was selected as follows: Figure 1 As shown, from the sequence comparison results of the representative strains of the three poxviruses, it can be seen that when the extension primer is designed before the mutated base, the extended base of the goatpox virus is T, the extended base of the sheeppox virus is A, and the extended base of the bovine lumpy skin disease virus is G.

[0054] The sequences and molecular weight (Mass) information of the amplification primers, extension primers and products are shown in Table 1 above.

[0055] 2. Sample Nucleic Acid Extraction

[0056] Sample sources: The goat pox, sheep pox and bovine nodular dermatitis samples in the examples were all from clinical disease samples.

[0057] Nucleic acid extraction: Nucleic acid extraction was performed using the Viral DNA / RNA Extraction Kit 4.0 from Xi'an Tianlong Technology Co., Ltd., coupled with a fully automated nucleic acid extractor. After extraction, 60 μL of the nucleic acid eluate from the deep-well plate was transferred to a clean EP tube and labeled.

[0058] 3. Testing steps

[0059] 1. Multiplex PCR amplification reaction

[0060] Multiple PCR amplification reaction was performed using SEQ ID NO: 1-4 as the test sample, using LSDV viral nucleic acid as the positive control and ultrapure water as the negative control to obtain amplified products, wherein the reaction system is shown in Table 2:

[0061] Table 2 Multiplex PCR reaction system

[0062] Reagent component name factory Item No. / Specifications Dosage PCR reaction mixture Zhejiang Dipu Diagnostic Technology Co., Ltd. DPGPM 2.5 μL PCR enzyme solution Zhejiang Dipu Diagnostic Technology Co., Ltd. DPGPM 0.3μL Multiplex amplification primer mix - 5 μM of each 0.2μL

[0063] The multiplex amplification primer mixture contains amplification primers SEQ ID NOs: 1-4; the concentration of each primer is 5 μM.

[0064] The reaction procedure is shown in Table 3:

[0065] Table 3 Multiplex PCR reaction program

[0066]

[0067] The actual test results of the multiplex PCR amplification reaction showed that the two-step cycling method could meet the amplification requirements and effectively amplify the target fragments.

[0068] 2. Dephosphorylation reaction

[0069] To eliminate excess dNTPs after multiplex PCR amplification and ensure the accuracy of mass spectrometry detection of single-base extension, shrimp alkaline phosphatase (SAP) was used for treatment. The SAP digestion enzyme reaction system is shown in Table 4:

[0070] Table 4 Dephosphorylation reaction system

[0071] Reagents Ultrapure water SAP reaction buffer SAP enzyme mixture Multiplex PCR products Dosage 1.53μL 0.17μL 0.3μL 5μL

[0072] SAP enzyme and buffer were purchased from Zhejiang Dipu Diagnostics Technology Co., Ltd. The reaction conditions were set as incubation at 37°C for 40 minutes to allow SAP to remove excess dNTPs; the SAP enzyme was then inactivated at 85°C for 5 minutes, and the reaction was stored at 4°C.

[0073] 3. Single base extension reaction

[0074] The components were added in sequence according to the reaction system in Table 5, and the prepared reaction system was subjected to single base extension according to the reaction procedure shown in Table 6.

[0075] Table 5 Single base extension reaction system

[0076]

[0077] The extension primer mixture contains amplification primers SEQ ID NO: 5-6; the concentration of each primer is 10 μM.

[0078] Table 6 Single base extension reaction procedure

[0079]

[0080] 4. Resin desalting purification and MALDI-TOF mass spectrometry detection

[0081] In the 384 reaction plate, add 18 μL DEPC water to each well at the set position. Add all the above single-base extension products (theoretical value is 9 μL, the actual volume may be slightly less than 9 μL due to some loss during the reaction) to each well containing DEPC water. After mixing, apply a sealing film and centrifuge to evenly distribute the mixture at the bottom of the well. Ensure that the Assay file accurately contains the molecular weight information of all single-base extension primers and their extension products, and import it into the operating software of the mass spectrometer. Open the nucleic acid flight mass spectrometer deck, remove the sealing film of the 384 reaction plate, and place it in the designated position. Set 10 μL of resin to be added to each well. The instrument will automatically run the product desalting and purification steps, and perform automatic spotting and mass spectrometry detection.

[0082] The results are determined based on whether a known single-base extension primer undergoes extension and the appearance of a specific extension peak and base. Mass spectrometry analysis results for each target show two peak positions: the single-base extension primer and the single-base extension product. If the primer extension conversion rate is 100%, the single-base extension primer peak disappears. If a single peak appears at the molecular weight corresponding to any extension product of a target and the signal-to-noise ratio (SNR) is greater than 6, the target is considered positive; otherwise, it is considered negative. The interpretation rules are shown in Table 1 above.

[0083] 4. Test results:

[0084] In the LSDV sample detection, the mass spectrometry detection results of P32 and GPCR sites are as follows Figure 2 shown.

[0085] In the sample detection of GTPV, the mass spectrometry detection results of P32 and GPCR sites were as follows: Figure 3 shown.

[0086] In the sample detection of SPPV, the mass spectrometry detection results of P32 and GPCR sites were as follows: Figure 4 shown.

[0087] In the mixed sample detection of GTPV and LSDV, the mass spectrometry detection results of P32 and GPCR sites were as follows: Figure 5 shown.

[0088] The three virus samples of LSDV, GTPV and SPPV were mixed to simulate the mixed infection of the three viruses. The mass spectrometry detection results of P32 and GPCR sites were as follows: Figure 6 shown.

[0089] The results show that the statistical information of the extension product Mass value of each sample is shown in the following table, which is consistent with the original sample information:

[0090] Sample Information P32 GPCR Interpretation of results LSDV A G LSDV GTPV A T GTPV SPPV A A SPPV LSDV+GTPV A G+T LSDV+GTPV LSDV+GTPV+SPPV A A+G+T LSDV+GTPV+SPPV

[0091] Example 2 Specificity verification of the detection method, primer combination and kit provided by the present invention

[0092] The samples to be tested used in this embodiment are Orfvirus (ORFV), Influenza virus, Mycoplasma and Japanese encephalitis virus.

[0093] The nucleic acid extraction and detection steps are the same as in Example 1.

[0094] The mass spectrometry results of ORFV at P32 and GPCR sites are as follows Figure 7 The statistical information of the extension product Mass value of each sample is shown in the following table, which is consistent with the original sample information:

[0095] Sample Information P32 GPCR Interpretation of results Contagious pustular virus No extension product peak No extension product peak LSDV / GTPV / SPPV negative influenza virus No extension product peak No extension product peak LSDV / GTPV / SPPV negative Mycoplasma No extension product peak No extension product peak LSDV / GTPV / SPPV negative Japanese encephalitis virus No extension product peak No extension product peak LSDV / GTPV / SPPV negative

[0096] It can be seen that for virus samples that are not within the detection range, no extension product peaks were observed at the GPCR and P32 sites, which fully demonstrates that the detection method, primer combination and kit provided by the present invention have good specificity.

[0097] Example 3 Sensitivity Verification of the Detection Method, Primer Combination, and Kit Provided by the Present Invention

[0098] In this example, qPCR was used to quantify the copy number of three nucleic acid samples (LSDV / GTPV / SPPV). The initial quantification of each positive nucleic acid was 1×10 5 copies / mL, and then dilute it 10 times, with the dilution loading ranging from 1×10 5 The dilution levels ranged from 1 copy / mL to 1 copy / mL, for a total of 6 dilutions. The assay was performed according to the methods and steps in Example 1, with 3 replicates for each gradient.

[0099] Goatpox virus 10 copies / mL test results are as follows Figure 8As shown, the results of the bovine lumpy skin disease virus 10 copies / mL test are as follows Figure 9 shown.

[0100] The test results showed that when the three virus-positive nucleic acids were diluted to 10 copies / mL, some extension primers for the GPCR target were not completely consumed, indicating that the detection method had reached the critical detection limit at this point. At this point, the extension primers for the P32 universal gene were completely consumed, suggesting that the detection sensitivity of the P32 gene is slightly higher than that of the GPCR gene. However, when the nucleic acid was diluted to 1 copy / mL, no extension products were detected in any of the three virus replicates. Therefore, the minimum detection limit of the detection method, primer combination, and kit provided by the present invention is between 1 copy / mL and 10 copies / mL.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A kit, characterized in that: The kit includes a primer composition for identifying bovine lumpy skin disease virus, goat pox virus and sheep pox virus; the composition includes amplification primers and single-base extension primers, the amplification primers include SEQ ID NOs: 1-4, and the single-base extension primers include SEQ ID NOs: 5-6; the kit also includes: PCR amplification reaction reagents, dephosphorylation reaction reagents and single-base extension reaction reagents.

2. A MALDI-TOF mass spectrometry method for detecting bovine lumpy skin disease virus, goat pox virus and sheep pox virus, characterized in that: The detection method is for non-disease treatment and / or diagnosis, and uses the conserved region of the P32 gene and the variant region of the GPCR gene as dual targets for detection, and includes the following steps: (S1) performing a PCR amplification reaction on the sample to be tested using SEQ ID NOs: 1-4 as amplification primers to obtain amplified products; the PCR amplification reaction is a multiplex PCR amplification reaction, and the reaction procedure includes: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds, annealing and extension at 60°C for 30 seconds, for 45 cycles; and storage at 4°C; (S2) dephosphorylating the amplified product using alkaline phosphatase to obtain a dephosphorylated product; (S3) Using SEQ ID NO: 5-6 as a single base extension primer, single base extension is performed on the dephosphorylated product to obtain an extension product; the single base extension reaction procedure includes: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds, annealing at 52°C for 5 seconds, and extension at 80°C for 5 seconds, for 40 cycles; wherein annealing and extension are performed separately for 5 cycles; extension at 72°C for 3 minutes, and storage at 4°C. (S4) purifying the extension product; (S5) MALDI-TOF mass spectrometry is used to detect the molecular weight of the purified product obtained in step (S4), and bovine lumpish skin disease virus, goat pox virus, and sheep pox virus are identified based on the difference in molecular weight.

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