A primer-probe combination for detecting sheep poxviruses and its application

Abstract

This invention relates to the field of virus detection technology, specifically to a primer-probe combination for detecting sheep poxviruses and its application. The primer-probe combination can be used to simultaneously detect or differentiate field strains of bovine nodular dermatophyte virus (LSDV), vaccine strains of LSDV, goat poxvirus, and sheep poxvirus. This invention establishes a quadruple qPCR detection method, enabling the identification and typing of four pathogens—LSDV field strains, LSDV vaccine strains, GTPV, and SPPV—in a single system. Furthermore, the excellent specificity, sensitivity, and repeatability exhibited by this method provide reliable technical support for the differential diagnosis and epidemiological investigation of LSD.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, specifically to a primer-probe combination for detecting sheep poxviruses and its application. Background Technology

[0002] Lumpy skin disease (LSD) is an acute and subacute viral infectious disease of cattle, caused by the lumpy skin disease virus (LSDV) of the genus Capripoxvirus (CaPV) in the family Poxviridae. All breeds of domestic cattle are susceptible to LSD. Currently, there are no reports of natural LSD infection in goats or sheep. However, recent studies indicate that the range of susceptible animals is expanding, with giraffes, Indian antelopes, and yaks now facing the risk of LSD infection. The incidence rate of LSD is generally between 5% and 45%. Affected animals exhibit characteristic clinical symptoms such as fever, nodular lesions on the skin, mucous membranes, and internal organs. It affects coat quality, milk production, and reproductive performance to varying degrees, and in severe cases, can lead to death, with a mortality rate generally below 10%.

[0003] LSDV, along with Goatpox virus (GTPV) and Sheeppox virus (SPPV), constitutes the genus *CaPV* within the family Poxviridae. CaPVs are double-stranded DNA viruses. LSDV has a genome size of 151 kbp, with an A+T ratio as high as 73% in its nucleotide composition, containing 156 putative open reading frames (ORFs). The genomic nucleotide homology between LSDV and SPPV and GTPV is over 97%. This high genomic homology among CaPVs stems from…

[0004] The fact that these substances are inherently difficult to distinguish serologically, and that they can induce heterologous cross-protection, makes differential diagnosis of them extremely difficult.

[0005] Traditional molecular biology detection methods are insufficient for effectively identifying four pathogens: LSDV field strains, LSDV vaccine strains, GTPV, and SPPV. Furthermore, LSD attenuated or heterologous attenuated vaccines may present problems in practical control due to adverse reactions or insufficient protective efficacy after vaccination. Therefore, establishing a stable and effective differential diagnostic method is crucial for monitoring LSD outbreaks, evaluating vaccine efficacy, and precisely controlling the epidemic. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings by providing a primer-probe combination for detecting LSDV viruses and its application. A quadruple qPCR detection method has been established, enabling the identification and typing of four pathogens—LSDV field strains, LSDV vaccine strains, GTPV, and SPPV—in a single system. Furthermore, the excellent specificity, sensitivity, and repeatability exhibited by this method provide reliable technical support for the differential diagnosis and epidemiological investigation of LSD.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A primer-probe combination for detecting sheep poxviruses, the primer-probe combination being capable of simultaneously detecting or differentiating field strains of bovine nodular dermatovirus, vaccine strains of bovine nodular dermatovirus, goat poxvirus, and sheep poxvirus.

[0009] The sequence information of the primer pairs and probe sets is as follows:

[0010] The forward primer for detecting CaPV (capoxvirus) is SEQ ID NO.1.

[0011] The reverse primer for detecting CaPV (capoxvirus) is SEQ ID NO.2.

[0012] The probe used to detect the field strain of bovine nodular dermatitis virus (LSDV) has the sequence SEQ ID NO.3;

[0013] The probe used to detect the bovine nodular dermatitis virus vaccine strain LSDV-VA has the sequence SEQ ID NO.4;

[0014] The probe used to detect goatpox virus GTPV has the sequence SEQ ID NO.5;

[0015] The probe used to detect sheep pox virus SPPV has the sequence SEQ ID NO.6.

[0016] Preferably, the 5′ and 3′ ends of the probe are labeled with fluorescein.

[0017] Preferably, the primer probes target the single nucleotide polymorphism (SNP) sites in the LSDV genome for the detection of the four viruses.

[0018] Preferably, the SNP site is located in the ORF 080 gene region of LSDV.

[0019] Preferably, the probe is a TaqMan MGB probe.

[0020] The use of a primer-probe combination for detecting sheep poxviruses in the preparation of formulations or kits for detecting or identifying sheep poxviruses.

[0021] Preferably, a kit for detecting sheep poxviruses comprises the primer pairs and probe sets described in any one of the above descriptions.

[0022] Preferably, the kit further comprises one or more of the following: 2X Pro Taq HSProbe Premix III, a positive control, and a negative control, required for real-time quantitative PCR.

[0023] The negative control was RNase-free water, and the positive controls were the constructed positive standard plasmids PUC57-LSDV080, PUC57-LSDV 080-VA, PUC57-GTPV 080, and PUC57-SPPV 080.

[0024] Preferably, the real-time quantitative PCR reaction program is: 95 ℃ for 30 s; 95 ℃ for 5 s, 60 ℃ for 30 s, for 45 cycles.

[0025] Preferably, the threshold setting principle is as follows: if the threshold line just exceeds the highest point of the amplification curve of the negative control, and the sample to be tested has no Ct value and no amplification curve, the result is judged as negative; if the Ct value of the sample to be tested is ≤40.0 and a typical amplification curve appears, the result is judged as positive. Specifically, if the signal is FAM, it is judged as positive for LSDV field strain nucleic acid; if the signal is HEX, it is judged as positive for LSDV vaccine strain nucleic acid; if the signal is ROX, it is judged as positive for GTPV nucleic acid; and if the signal is Cy5, it is judged as positive for SPPV nucleic acid.

[0026] The beneficial effects of this invention are:

[0027] This invention establishes a quadruple qPCR detection method for CaPVs.

[0028] High specificity: This method utilizes primers and probes designed for specific SNP sites, which can effectively amplify and distinguish LSDV field strains, LSDV vaccine strains, GTPV and SPPV, and has no cross-reaction with other common bovine and ovine viruses.

[0029] High sensitivity: This method has excellent detection sensitivity and can detect trace amounts of viral nucleic acid.

[0030] Highly efficient and economical: A single qPCR reaction can achieve simultaneous differential diagnosis of four antigens, significantly improving detection efficiency and reducing detection costs and sample consumption.

[0031] High concordance rate: Clinical samples have verified that the concordance rate between this method and the national standard qPCR detection method can reach 98.7%, demonstrating good reliability and practical value, and providing reliable technical support for the differential diagnosis, epidemiological investigation and epidemic prevention and control of LSD. Attached Figure Description

[0032] Figure 1 These are the SNP sites targeted by the probe of this invention;

[0033] Figure 2 The present invention comprises the PCR amplification of the target gene fragment and the linearized PUC57 vector; wherein, M is DNAMaker; 1 is the LSDV 080 gene fragment; 2 is the LSDV 080-VA gene fragment; 3 is the GTPV 080 gene fragment; 4 is the SPPV 080 gene fragment; and 5 is the linearized PUC57 vector.

[0034] Figure 3 This is the map of the CaPVs positive plasmid constructed in this invention;

[0035] Figure 4 This is a specificity test of the quadruple qPCR method of the present invention; wherein, A is the amplification curve of PUC57-LSDV 080 plasmid; B is the amplification curve of PUC57-LSDV 080-VA plasmid; C is the amplification curve of PUC57-GTPV 080 plasmid; and D is the amplification curve of PUC57-SPPV 080 plasmid.

[0036] Figure 5 This is a sensitivity test of the quadruple qPCR method of the present invention; wherein, A is the detection limit of LSDV field strain; B is the detection limit of LSDV vaccine strain; C is the detection limit of GTPV; and D is the detection limit of SPPV. Detailed Implementation

[0037] Main reagents

[0038] 2×Phanta® Flash Master Mix (P510, Nanjing Novizan Biotechnology Co., Ltd.); QuickCut™ EcoR I and QuickCut™ Hind III (1611 and 1615, Baori Biotech (Beijing) Co., Ltd.); ClonExpress Ultra One Step Cloning Kit (C115, Nanjing Novizan Biotechnology Co., Ltd.); Agarose Gel DNA Recovery Kit, Rapid Plasmid Mini-Prep Kit (DP219, DP105, Tiangen Biotech (Beijing) Co., Ltd.); GL DNA Marker 2000, GL DNA Marker 5000 and AG-CelRed Nucleic Acid Gel Dyes (AG11904, AG11906 and AG11918, Hunan Aikerui Biotechnology Co., Ltd.); EasyScript cDNA First-Strand Synthesis Kit (AE301-03, Beijing TransGen Biotech Co., Ltd.).

[0039] Main instruments and equipment

[0040] Gene amplification instrument (Hangzhou Bori Technology Co., Ltd.); Constant temperature shaking incubator (MQL-61HR, Shanghai Minquan Instrument Co., Ltd.); Gel imaging system (G Box, Syngene); Benchtop high-speed refrigerated centrifuge (Fresco21, ThermoFisher); Clean bench (BCM-1000A, Suzhou Antai Air Technology Co., Ltd.); Vertical pressure steam sterilizer (YXQ-100G, Shanghai Boxun Medical Bio-Instrument Co., Ltd.); Medical refrigerator (HYC-310, Qingdao Haier Co., Ltd.); Vortex mixer (MIX2000, Hangzhou Ruicheng Instrument Co., Ltd.); Nucleic acid electrophoresis system (JY-SPCT, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.); Blue light gel cutter (UV-Cut106, Primacy Instruments (Hangzhou) Co., Ltd.); Ultra-micro UV spectrophotometer (NanoDrop one, Thermo); Real-time PCR instrument (LightCycler). 96, Roche); constant temperature water bath, forced air drying oven, electric constant temperature incubator (DK-500S, DHG-9140A, DNP-9082, Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0041] Example 1

[0042] Final list of primer and probe sequences.

[0043] Based on the SNP site region at ORF 080 of LSDV field strains, LSDV vaccine strains, GTPV, and SPPV, primer pairs and probe sets were designed using PrimerPremier and SnapGene software, taking into account basic parameters such as primer and probe sequence length, annealing temperature (Tm value), and GC content. Their sequence number information is shown in Table 1 below, and the target SNP sites of the probes are as follows: Figure 1 As shown.

[0044] Table 1. Serial number information of primer pairs and probe sets

[0045] Example 2

[0046] Construction process and identification results of positive standard plasmids.

[0047] Construction of positive standard plasmids

[0048] Using the viral genomes of LSDV field strain, GTPV, and SPPV as templates, gene fragments of the three viruses at the gene detection target sites in ORF 080 were amplified using homologous recombination primers 080-PUC57-F and 080-PUC57-R. The band size in the gel image was approximately 200 bp, consistent with the theoretical value. Figure 2 Then, using the gene fragment at the target location of the LSDV field strain as a template, the gene fragment at the target location of the LSDV vaccine strain was amplified using point mutation primers 080-P-VA-F and 080-P-VA-R, respectively, combined with homologous recombination primers 080-PUC57-F and 080-PUC57-R. The band size in the nucleic acid electrophoresis gel image was 200 bp, consistent with the theoretical value. Figure 2 Finally, the PUC57 E. coli cloning vector was linearized by double digestion with EcoRI and Hind III. The band size in the nucleic acid electrophoresis gel image was 2600 bp, which is consistent with the theoretical value. Figure 2 ).

[0049] Homologous recombination was used to ligate the target gene fragment obtained above with the linearized PUC57 vector to construct qPCR recombinant positive plasmids. After transformation into Fast-T1 competent cells, single clones were selected for sequencing analysis. Plasmids with correct sequencing alignment were used as positive standard plasmids for subsequent qPCR experiments. Successfully constructed qPCR positive standard plasmids include: PUC57-LSDV 080, PUC57-LSDV 080-VA, PUC57-GTPV 080, and PUC57-SPPV 080. The plasmid maps are shown below. Figure 3 .

[0050] The specific process is as follows:

[0051] Primer design and synthesis

[0052] To construct a positive standard plasmid for qPCR, new primers were designed before the upstream primer and after the downstream primer of the original qPCR primers. Homologous arms of the gene sequence of the linearized plasmid obtained by double digestion of the PUC57 plasmid with EcoRI and HindIII were added to the 5' end of each of the newly designed upstream and downstream primers (Table 2). The target gene amplified by these primers can be ligated with the PUC57 linearized vector via homologous recombination to construct a recombinant positive standard plasmid.

[0053] Table 2 Primers used for constructing qPCR positive standard plasmids

[0054] Note: The bolded sequences are homologous sequences of the PUC57 plasmid.

[0055] Point mutation primers were designed based on the LSDV vaccine strain genome sequence. Point mutations were performed using the gene sequence of the target region of the LSDV field strain as a DNA template to obtain the corresponding gene fragments of the LSDV vaccine strain (Table 3).

[0056] Table 3. Point mutation primers used to construct positive control plasmids

[0057] Note: Bold bases are point mutation bases.

[0058] Target gene amplification

[0059] The viral genomes of LSDV, GTPV and SPPV were obtained using a viral DNA / RNA extraction kit. The PCR reaction system was prepared using 2×Phanta® Flash Master Mix high-fidelity enzyme premix. The components and volumes are shown in Table 4.

[0060] Table 4. Preparation of PCR reaction system

[0061] PCR reaction program: 98℃ for 30 s; 98℃ for 10 s, 56℃ for 5 s, 72℃ for 10 s, 30 cycles; 72℃ for 1 min. Prepare a 1% nucleic acid agarose gel (with 1 / 10000 nucleic acid gel dye). After PCR amplification, perform nucleic acid electrophoresis (160 V for 20 min), and acquire images on a gel imaging system. Precisely cut the gel containing the target gene fragment from the blue light gel cutter's field of view. Purify and recover the target gene using an agarose gel DNA recovery kit according to the manufacturer's instructions. Determine the concentration and purity of the target gene using a NanoDrop micro spectrophotometer.

[0062] The point mutation of the target region gene sequence for LSDV field strain detection was converted to the target region gene sequence for LSDV vaccine strain detection via overlap PCR. The specific procedure is as follows:

[0063] The first round of PCR used the gene sequence of the target region of the LSDV field strain ORF 080 as a DNA template. Two sets of primers, 080-PUC57-F and 080-P-VA-R and 080-P-VA-F and 080-PUC57-R, were used for PCR amplification. The gene sequence of the target region of the LSDV field strain ORF 080 was amplified into two gene fragments, and nucleotide mutation sites were introduced.

[0064] In the second round of PCR, the upper and lower gene fragments of the target region gene sequence of the LSDV field strain ORF 080 obtained in the previous step were added to the same reaction system as a DNA template, using a primer set of 080-PUC57-F and 080-PUC57-R. Overlap PCR was then used to ligate the two fragments after the point mutation, forming the complete LSDV vaccine strain ORF080 target region gene sequence. Nucleic acid electrophoresis and agarose gel DNA recovery and purification were performed as described above. The concentration and purity of the target gene were determined using a NanoDrop micro-spectrophotometer.

[0065] Vector double enzyme digestion

[0066] (1) Fast-T1 glycerol bacteria containing PUC57 plasmid were inoculated into LB medium containing ampicillin and placed in a constant temperature shaking incubator at 37℃ and 200 r / min for activation and overnight proliferation.

[0067] (2) Centrifuge at 12000 r / min for 2 min to collect the bacterial pellet, and extract the PUC57 plasmid using a rapid plasmid mini-extraction kit according to the manufacturer's instructions.

[0068] (3) The concentration and purity of PUC57 plasmid were determined using a NanoDrop micro spectrophotometer.

[0069] (4) Prepare the EcoRI and HindIII double enzyme digestion reaction system (Table 5) and digest it at 37℃ for 2 h.

[0070] Table 5. Preparation of reaction system for plasmid digestion

[0071] (5) Nucleic acid electrophoresis and agarose gel DNA recovery and purification were performed according to the kit instructions.

[0072] Recombinant plasmid construction

[0073] The ligation of the PUC57 linearized empty vector and the target gene was performed using homologous recombination. Referring to the ClonExpressUltra One Step Cloning Kit manual, the volumetric amounts of each component in the reaction system were calculated based on the concentrations of the PUC57 linearized empty vector and the target gene. The homologous recombination system was prepared on ice and incubated in a 50°C water bath for 30 min, followed by subsequent transformation experiments on ice. The transformation of the recombinant PUC57 plasmid into Fast-T1 cloning chemocompetent cells was performed as follows:

[0074] (1) Take Fast-T1 cloned chemical competent cells out of the -80℃ ultra-low temperature freezer and place them on ice to thaw.

[0075] (2) Take 3 μL of recombinant PUC57 plasmid and gently add it to competent cells. Gently tap the tube wall to mix (do not blow or shake vigorously to mix), and let stand on ice for 30 min.

[0076] (3) After placing competent cells in a 42°C water bath for 45 s heat shock, immediately place them on ice to cool for 2-3 min.

[0077] (4) Add 600 μL of LB liquid culture medium (antibiotic-free) and activate at 37°C and 200 r / min for 1 h.

[0078] (5) Take out the ampicillin-resistant LB solid medium plates and preheat them in a 37°C incubator.

[0079] (6) Centrifuge the activated Fast-T1 bacterial culture at 5000 r / min for 5 min and discard 600 μL of supernatant. Resuspend the bacterial culture in the remaining culture medium, add it to the surface of the solid culture medium plate and spread it evenly with a sterile spreader.

[0080] (7) Place the solid culture medium plate in a 37°C incubator and invert it for 12-16 h. Select single colonies that are round and uniform in size and culture them overnight.

[0081] (8) A portion of the bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing analysis. Positive plasmid strains with correct sequencing were selected and preserved with 50% glycerol solution at −20℃.

[0082] Preparation of positive plasmid standards

[0083] (1) The positive plasmid strain was activated in LB medium containing ampicillin and cultured overnight.

[0084] (2) Centrifuge at 12000 r / min for 2 min to collect the bacterial pellet, and extract the positive plasmid using a rapid plasmid mini-extraction kit according to the manufacturer's instructions.

[0085] (3) The concentration and purity of positive plasmids were determined using a NanoDrop micro spectrophotometer.

[0086] (4) First, calculate the molecular weight of each positive plasmid according to the formula: MW plasmid (Da) = plasmid size (bp) × 660 Da / bp.

[0087] (5) Then, according to the formula: copy number (copies / μL) = ((plasmid concentration (ng / μL) × 10−9) / MW plasmid) × 6.022 × 1023 copies / mol, calculate the copy number concentration of each positive plasmid.

[0088] (6) The copy number of each positive plasmid was uniformly reduced to 107 copies / μL by dilution.

[0089] (7) Then, by serially diluting by 10-fold, positive plasmid standards with 107-101 copies / μL were obtained.

[0090] Specificity test

[0091] Viral genomes of BRV, BVDV, PPRV, and BCoV were extracted using a viral DNA / RNA extraction kit. Viral RNA extraction was performed according to the manufacturer's instructions. The genomic RNA of each virus was reverse transcribed into cDNA using the EasyScript cDNA first-strand synthesis kit. The RNA reverse transcription reaction system was prepared according to Table 6.

[0092] Table 6 RNA reverse transcription system

[0093] Reverse transcription program: 42℃ for 30 min, 85℃ for 5 s.

[0094] The above-mentioned viral cNDA and positive plasmid standards of LSDV field strain, LSDV vaccine strain, GTPV, and SPPV were used as nucleic acid samples to verify the specificity of the qPCR method in common bovine and ovine pathogens in clinical practice.

[0095] The preparation of the reaction system and the reaction procedure are shown in Table 7.

[0096] Table 7. Preparation of reaction system for quadruple quantitative PCR

[0097] Two-step qPCR reaction program: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, 45 cycles.

[0098] Example 3

[0099] The components, concentrations, and optimized parameters of the amplification program for the quadruple qPCR reaction system.

[0100] Optimize reaction conditions

[0101] While keeping other variables constant during the qPCR reaction, the annealing temperature, probe concentration, and primer concentration were changed sequentially. The optimal reaction system and procedure were determined based on the CT value of the positive plasmid standards. The optimization results of the annealing temperature conditions showed that, within the annealing temperature gradient of 56℃ to 63℃, the CT values ​​of all four positive plasmid standards were lowest when the annealing temperature was 60℃ (Table 8).

[0102] Table 8 Optimization of Annealing Temperature Conditions

[0103] The results of primer concentration optimization showed that by changing the amount of primers added to the reaction system, the primer concentration in the system was adjusted, and the CT values ​​of the four positive plasmid standards were the lowest when the primer addition amount was 1.00 μL (Table 9).

[0104] Table 9 Primer Concentration Conditions Optimization

[0105] Optimization results of probe concentration conditions showed that by changing the amount of probe added to the reaction system, the probe concentration in the system was adjusted. When the addition volume of 080-LSDV-P and 080-LSDV-VA-P was 0.6 μL, the CT values ​​of the two positive plasmid standards PUC57-LSDV 080 and PUC57-LSDV 080-VA were the lowest. When the addition volume of the 080-GTPV-P probe was 0.5 μL, the CT value of PUC57-GTPV 080 was the lowest. When the addition volume of the 080-SPPV-P probe was 0.4 μL, the CT value of PUC57-SPPV080 was the lowest (Table 10).

[0106] Table 10 Optimization of probe concentration conditions

[0107] In summary, the optimal reaction system and procedure are determined as follows:

[0108] Table 11 Optimal reaction system for quadruple real-time PCR

[0109] The optimal qPCR reaction program is: 95 ℃ for 30 s; 95 ℃ for 5 s, 60 ℃ for 30 s, for 45 cycles.

[0110] Example 4

[0111] Specific data from specificity, sensitivity (limit of detection), and repeatability (intra-batch / inter-batch) experiments conducted on the optimized method.

[0112] Specificity test

[0113] Viral genomes of common bovine and ovine pathogens (BRV, BVDV, PPRV, BCoV) were extracted and reverse transcribed to serve as test samples. Quadruple qPCR detection was performed using positive standard plasmids PUC57-LSDV 080, PUC57-LSDV 080-VA, PUC57-GTPV 080, and PUC57-SPPV080 as positive controls to investigate the specificity of this method in clinical applications. The results showed that this method could not effectively amplify the viral genomes of BRV, BVDV, PPRV, and BCoV, and no cross-reactivity was observed. The primers and probes exhibited good specificity, and in clinical applications, it can achieve specific differential diagnosis of LSDV field strains, LSDV vaccine strains, GTPV, and SPPV. Figure 4 ).

[0114] Sensitivity test

[0115] Positive plasmid standards for LSDV field strains, LSDV vaccine strains, GTPV, and SPPV were serially diluted 10-fold to obtain positive plasmid standards with concentration gradients ranging from 10⁷ to 10¹ copies / μL. The optimized reaction system and procedure were used to perform qPCR amplification on the positive plasmid standards of the four strains to study the sensitivity of the method. The results showed that the detection limit of the LSDV quadruple qPCR method for all four strains reached 10 copies / μL. Figure 5 )

[0116] Repeatability test

[0117] The LSDV quadruple qPCR detection method was used to perform intra-assay and inter-assay repeatability tests on positive plasmid standards at three concentrations (10⁶ copies / μL, 10⁴ copies / μL, and 10² copies / μL) corresponding to LSDV field strain, LSDV vaccine strain, GTPV, and SPPV. The results showed that the intra-assay coefficient of variation (COP) for the four positive plasmid standards ranged from 0.44% to 2.24%, and the inter-assay COP ranged from 0.94% to 2.81%. The overall COP was less than 3%, indicating that the detection method has good repeatability (Table 12).

[0118] Table 12 Repeatability tests of the quadruple qPCR method

[0119] Example 5

[0120] Comparative data and concordance rate analysis of parallel testing of 156 clinical samples using this method and existing national standard methods (98.7% in the example).

[0121] Clinical sample testing

[0122] 156 clinical samples were tested using the qPCR method in the national standard for diagnosis of bovine nodular dermatitis (GB / T 39602-2020) and the qPCR method established in this study. The results showed that the quadruple qPCR method detected LSDV in 21 field-grown samples, GTPV in 98 samples (including 2 LSDV field-grown samples), and was negative in 39 samples. The national standard qPCR method detected LSDV in 19 samples and was negative in 137 samples (Table 13). In summary, the overall concordance rate for LSDV detection between the two qPCR methods was 98.7%, indicating that the established quadruple qPCR method has good applicability in clinical sample testing.

[0123] Table 13 Clinical Sample Detection Results

[0124] The specific embodiments described herein are merely illustrative examples of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the scope defined by this invention.

Claims

1. A primer-probe combination for detecting sheep poxviruses, characterized in that: The primer-probe combination can be used to simultaneously detect or differentiate bovine nodular dermatovirus field strains, bovine nodular dermatovirus vaccine strains, goatpox virus, and sheeppox virus. The sequence information of the primer pairs and probe sets is as follows: The forward primer for detecting CaPV (capoxvirus) is SEQ ID NO.

1. The reverse primer for detecting CaPV (capoxvirus) is SEQ ID NO.

2. The probe used to detect the field strain of bovine nodular dermatitis virus (LSDV) has the sequence SEQ ID NO.3; The probe used to detect the bovine nodular dermatitis virus vaccine strain LSDV-VA has the sequence SEQ ID NO.4; The probe used to detect goatpox virus GTPV has the sequence SEQ ID NO.5; The probe used to detect sheep pox virus SPPV has the sequence SEQ ID NO.

6.

2. The primer-probe combination for detecting sheep poxviruses according to claim 1, characterized in that, The probe was labeled with fluorescein at its 5′ and 3′ ends.

3. The primer-probe combination for detecting sheep poxviruses according to claim 1, characterized in that, The primer probes target the single nucleotide polymorphism (SNP) sites in the genome of the four viruses.

4. The primer-probe combination for detecting sheep poxviruses according to claim 2, characterized in that, The SNP site is located in the ORF 080 gene region of the virus.

5. The primer-probe combination for detecting sheep poxviruses according to claim 4, characterized in that, The probe is a TaqMan MGB probe.

6. The use of the primer-probe combination for detecting sheep poxviruses according to any one of claims 1-5 in the preparation of a formulation or kit for detecting or identifying sheep poxviruses.

7. The kit for detecting sheep poxviruses according to claim 6, characterized in that, It comprises the primer pair and probe set as described in any one of claims 1-5.

8. The kit for detecting sheep poxviruses according to claim 7, characterized in that, The kit also contains one or more of the following: 2X Pro Taq HS Probe Premix III, a positive control, and a negative control, required for real-time quantitative PCR. The negative control was RNase-free water, and the positive controls were the constructed positive standard plasmids PUC57-LSDV080, PUC57-LSDV 080-VA, PUC57-GTPV 080, and PUC57-SPPV 080.

9. A kit for detecting sheep poxviruses according to claim 8, characterized in that, The real-time quantitative PCR reaction program was: 95 ℃ for 30 s; 95 ℃ for 5 s, 60 ℃ for 30 s, for 45 cycles.

10. A kit for detecting sheep poxviruses according to claim 8, characterized in that, The threshold setting principle is as follows: if the threshold line just exceeds the highest point of the amplification curve of the negative control, and the test sample has no Ct value and no amplification curve, the result is judged as negative; if the test sample has a Ct value ≤ 40.0 and shows a typical amplification curve, the result is judged as positive. Specifically, a FAM signal indicates positive nucleic acid of LSDV field strain; a HEX signal indicates positive nucleic acid of LSDV vaccine strain; a ROX signal indicates positive nucleic acid of GTPV; and a Cy5 signal indicates positive nucleic acid of SPPV.

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