MALDI-TOF mass spectrum detection method for bovine nodular skin disease virus, goat pox virus and sheep pox virus

Abstract

The invention relates to the field of pathogenic microorganism detection, in particular to an MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometric detection method for bovine nodular skin disease virus, goat pox virus and sheep pox virus and a primer combination used by the method. According to the present invention, the multiple PCR amplification technology, the double-gene target strategy, the single base extension reaction and the high precision analysis ability of the MALDI-TOF MS are combined, and the efficient method capable of simultaneously carrying out multi-target precise detection on the three sheep pox viruses is provided. According to the invention, two pairs of specific amplification primers are adopted for targeted amplification of a target gene segment, two extension primers are supplemented for subsequent single-base extension reaction, and molecular weight difference generated by a single base in an extension product on MALDI-TOF MS is accurately measured, so that the detection of the single-base extension reaction in a single-tube reaction system can be realized. According to the method, whether a sample is infected by the three sheep pox viruses or not can be efficiently identified, and meanwhile, one, several or all of the three viruses which are specifically infected can be further subdivided and clearly reported, so that the comprehensiveness, specificity and accuracy of detection are greatly improved.

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 dermatosis 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 genus Capripoxvirus (CaPV). Infection with this genus of viruses can cause fever, skin or mucosal nodules (or papules), lymphadenopathy and other symptoms. Diseases caused by poxviruses can significantly reduce production performance, and animals with severe disease can also die. For example, lumpy skin disease in cattle can cause deep holes in the skin of infected cattle, making the skin unusable, male animals infertile, female animals aborted, and dairy cows' milk production significantly reduced, and in severe cases, death. When goatpox and sheeppox viruses infect extremely susceptible sheep breeds, the mortality rate can be as high as 100%. The epidemics of the three diseases have caused huge economic losses worldwide. Due to the economic importance of cattle, sheep and goat farming and the potential rapid cross-border spread of the virus, lumpy skin disease in cattle, goatpox and sheeppox are all listed as notifiable diseases by the World Organization for Animal Health (OIE).

[0003] At present, the classic virological separation technology and serological analysis strategy are usually used for the differential diagnosis of bovine nodular dermatosis, goat pox and sheep pox. The core of the virus isolation technology is to collect suspected lesion tissue samples, proliferate the virus through a cell culture system, and then use electron microscopy technology to finely analyze the morphological and structural characteristics of the virus particles obtained by culture. Although this strategy can accurately identify the type and subtype of the virus, its significant disadvantage is that it is time-consuming (often taking several days to several weeks), the operation process is complicated, and the professional skills of the experimenters are strict, which restricts its popularization and application in clinical practice and large-scale screening. Because LSDV, GTPV and SPPV strains show a high degree of similarity at the antigenic level, it is difficult for traditional serological methods to effectively distinguish the two, but there are differences in the gene sequences of LSDV, GTPV and SPPV strains. In view of this, PCR technology, with its advantages of simple operation, fast detection speed, high sensitivity and strong specificity, has shown great potential in the 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 the early diagnosis of the disease. However, the PCR method is generally limited to the detection of a single or a few viruses, and it is difficult to achieve comprehensive coverage and accurate typing of the virus spectrum. In addition, although high-throughput sequencing (HTS) technology can achieve accurate identification of viruses, its high cost, complex operating procedures and relatively long data processing time all constitute obstacles to its widespread application in the field of rapid diagnosis.

[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 nodular 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 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) with high-precision analysis capabilities, aiming to develop an efficient method that can simultaneously perform multi-target accurate detection of three sheep pox viruses. Specifically, the present invention designs and applies two pairs of specific amplification primers to target amplify target gene fragments, and is supplemented with two extension primers for subsequent single base extension reactions, and 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. Not only can it be efficiently identified whether there are three sheep pox viruses (i.e., LSDV, GTPV, and SPPV) in the sample, but it can also be further subdivided and clearly reported 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 in this way:

[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 NO: 1-4, and the single-base extension primers comprising SEQ ID NO: 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 conservative in the capripoxvirus genus, but there are also sufficient sequence differences to distinguish different types of viruses. Based on the conservative 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 conservative sequence region in the amplification region. At the 3' end of the single-base extension primer, a designed and determined base is allowed to be extended as a specific sequence marker for different capripox viruses. 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 in the position before the mutant base, the extension base of the goat poxvirus is T, the extension base of the sheep poxvirus is A, and the extension 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] Further, 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.

[0013] The kit also includes a dephosphorylation reagent. In some embodiments, the components of the dephosphorylation reagent include: alkaline phosphatase and buffer. Further, 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 detection method for bovine nodular dermatitis virus, goat pox virus and sheep pox virus, wherein the detection method is for non-disease treatment and / or diagnosis, and the conservative region of the P32 gene and the variable region of the GPCR gene are used as dual targets for detection, and the method comprises the following steps:

[0016] (S1) using SEQ ID NO: 1-4 as amplification primers, performing PCR amplification reaction on the sample to be tested to obtain an amplification 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 the 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 another 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 lumpish dermatitis 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 judged based on whether the single-base extension primer is extended and a specific extension peak (i.e., a peak matching the expected extension product molecular weight) and the base appear.

[0026] The mass spectrometry analysis results correspond to two peak positions, namely, 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 lower than 6, it is judged to be negative.

[0027] The sequence and molecular weight (Mass) information of the amplification primer, extension primer and product are shown in Table 1 below:

[0028] Table 1

[0029]

[0030] The shaded part of the possible extension product sequence is the extension base. In the interpretation rule, A / G / T represent different extension bases. LSDV, GTPV and SPPV correspond to bovine lumpish 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 uses two pairs of specific amplification primers, which are intended to target and amplify the target gene fragment, and are supplemented with two extension primers to support the subsequent single-base extension reaction. By using MALDI-TOF mass spectrometry to accurately measure the molecular weight difference caused by a single base in the extension product, this method can efficiently identify whether there is infection with three different viruses in the genus Capripoxvirus (specifically, bovine nodular skin disease virus LSDV, goat pox virus GTPV, and sheep pox virus SPPV) in a single reaction system, and further specifically type the infected viruses, thereby significantly improving the comprehensiveness, specificity and accuracy of the detection.

[0033] The present invention demonstrates accurate typing capabilities, and the interpretation rules are concise and efficient. By optimizing the selection of amplification sites and the addition of modified bases, this method ensures that the molecular weight of the extended probe is effectively distinguished within the detection range, achieving the goal of multiple detection, and the ability of each well reaction to support multiple detection far exceeds that of traditional fluorescent quantitative PCR methods. In addition, this method does not need to rely on fluorescent dyes, gets rid of the limitations of fluorescent channels, and improves the flexibility and accuracy of detection.

[0034] In order to improve the detection efficiency and throughput, the present invention is compatible with high-density 384-well chips, so that single-well reactions can complete all experiments and data analysis in a relatively short time, which is suitable for rapid screening of large-scale samples. At the same time, the sample loading process is flexible and the chip can be used multiple times, further reducing costs. Researchers can adjust the detection reagents according to actual needs, and the operation is simple and does not require complex professional guidance, which enhances 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 creative work.

[0036] Figure 1 A schematic diagram of the alignment of GPCR gene sequences 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 the bovine lumpy skin disease virus (LSDV) sample;

[0038] Figure 3 This is the MALDI-TOF mass spectrometry test result of goat pox 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 mixed samples of bovine lumpy skin disease virus (LSDV) and goat pox virus (GTPV);

[0041] Figure 6 The results of MALDI-TOF mass spectrometry for mixed samples 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 test result of goatpox virus 10 copies / mL;

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

[0045] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The methods used in the following examples are all conventional methods unless otherwise specified. 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 various biological materials described in the examples are only provided as an experimental method to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0048] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or equipment 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 equipment. 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: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the previously associated objects 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 nodular 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 dermatitis virus, goat pox virus and sheep pox virus were downloaded from the GenBank database after complete annotation as reference sequences, and P32 and GPCR genes were selected as dual targets.

[0052] Among them, 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 virus species.

[0053] After sequence alignment and phylogenetic tree analysis, the conserved region of the P32 gene was confirmed, and the mutation site of the GPCR gene was selected. According to the conserved sequence of the selected target gene, specific amplification primers were designed. A 10-base universal sequence 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 in 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 selection for the GPCR gene extension 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 in the position before the mutant base, the extension base of the goat poxvirus is T, the extension base of the sheep poxvirus is A, and the extension base of the bovine lumpy skin disease virus is G.

[0054] The sequence and molecular weight (Mass) information of the amplification primer, extension primer and product are shown in Table 1 above.

[0055] 2. Sample Nucleic Acid Extraction

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

[0057] Nucleic acid extraction: Use the viral DNA / RNA extraction kit 4.0 of Xi'an Tianlong Technology Co., Ltd. with a fully automatic nucleic acid extractor for nucleic acid extraction. After extraction, transfer 60 μL of nucleic acid eluate in the deep-well plate to a clean EP tube and mark it.

[0058] 3. Testing steps

[0059] 1. Multiplex PCR amplification reaction

[0060] Multiple PCR amplification reaction was performed on the sample to be tested using SEQ ID NO: 1-4, with LSDV viral nucleic acid as a positive control and ultrapure water as a 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. / Specification Dosage PCR reaction mixture Zhejiang Dipu Diagnostics Technology Co., Ltd. DPGPM 2.5μL PCR enzyme solution Zhejiang Dipu Diagnostics Technology Co., Ltd. DPGPM 0.3μL Multiplex Amplification Primer Mix - 5 μM of each 0.2μL

[0063] The multiple amplification primer mixture solution includes amplification primers SEQ ID NO: 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 cycle method can meet the amplification requirements and can effectively amplify the target fragment.

[0068] 2. Dephosphorylation reaction

[0069] In order to eliminate excess dNTPs after multiple PCR amplification reactions and ensure the accuracy of single-base extension detection by mass spectrometry, 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] The SAP enzyme and buffer were purchased from Zhejiang Dipu Diagnostic Technology Co., Ltd. The reaction conditions were set to incubate at 37°C for 40 min, allowing SAP to remove the remaining dNTPs; then the SAP enzyme was inactivated at 85°C for 5 min, and then stored at 4°C.

[0073] 3. Single base extension reaction

[0074] The components were added in sequence according to the reaction system sequence in Table 5, and the configured reaction system was used for 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 is amplification primers SEQ ID NO: 5-6; the concentration of each primer is 10 μM.

[0078] Table 6 Single base extension reaction program

[0079]

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

[0081] Pre-add 18μL DEPC water to each well at the set position in the 384 reaction plate, add all the above single base extension products (theoretical value 9μL, the actual volume may be slightly less than 9μL, there is a certain loss during the reaction) to each well containing DEPC water, mix well, stick the sealing film, and centrifuge to evenly distribute the mixture at the bottom of the well. Make sure 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 put it in the designated position. Set each well to add 10μL of resin, and the instrument will automatically run the product desalting and purification steps, and perform automatic spotting and mass spectrometry detection.

[0082] The result is determined based on whether the known single-base extension primer is extended and a specific extension peak and base appear. The mass spectrometry analysis results of each target 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 >6, the target is judged to be positive: otherwise, it is judged to be 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 test 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 Mass value of the extension products 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 each virus sample that is not within the detection range, no extension product peak is 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 dilutions ranged from 1 copy / mL to 1 copy / mL, with a total of 6 dilutions. The tests were performed according to the methods and steps in Example 1, and each gradient was repeated 3 times.

[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 Fig. 9 shown.

[0100] The test results show that when the three virus-positive nucleic acids were diluted to 10 copies / mL, some of the extension primers of the GPCR target were not completely consumed, indicating that the critical detection limit of the detection method has been reached at this time, while the extension primers of the P32 universal gene were completely consumed at this time. It is speculated that the detection sensitivity of the P32 gene is slightly higher than that of the GPCR gene, but when the nucleic acid was diluted to 1 copy / mL, no extension products were detected in each duplicate well of the three viruses. 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 protection scope of the present invention.

Claims

1. A primer composition for identifying bovine lumpish skin disease virus, goat pox virus and sheep pox virus, characterized in that: The composition includes an amplification primer and a single-base extension primer, wherein the amplification primer includes SEQ ID NOs: 1-4, and the single-base extension primer includes SEQ ID NOs: 5-6.

2. The use of the primer composition according to claim 1, characterized in that: The application is non-disease treatment and / or diagnosis, and the primer composition is used for at least one of the uses (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.

3. A kit, characterized in that: The kit comprises the primer combination according to claim 1. The kit according to claim 3 , further comprising a PCR amplification reaction reagent. The kit according to claim 3 , further comprising a dephosphorylation reaction reagent. The kit according to claim 3 , further comprising a single base extension reaction reagent.

7. A MALDI-TOF mass spectrometry method for detecting bovine nodular 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 conservative region of the P32 gene and the variable region of the GPCR gene as dual targets for detection, and includes the following steps: (S1) using SEQ ID NO: 1-4 as amplification primers, performing PCR amplification reaction on the sample to be tested to obtain an amplification product; (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, performing single base extension on the dephosphorylated product to obtain an extension product; (S4) purifying the extension product; (S5) MALDI-TOF mass spectrometry detection.

8. The detection method according to claim 7, characterized in that: In the step (S1), 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, 45 cycles; and storage at 4°C.

9. The detection method according to claim 7, characterized in that: In the step (S3), the reaction procedure of single base extension includes: 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, 40 cycles; annealing and extension are performed separately for 5 cycles; extension at 72°C for 3min, and storage at 4°C.

10. The detection method according to claim 7, characterized in that: In the step (S5), the molecular weight of the purified product obtained in step (S4) is detected by MALDI-TOF mass spectrometry, and bovine nodular dermatosis virus, goat pox virus and sheep pox virus are identified based on the difference in molecular weight.

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