Dual-fluorescence PCR primers, probes, method and kit for identifying capripoxvirus and lumpy skin disease virus

[0045]Example 1

[0046]This set of primer probes is designed for all members of the genus goat pox virus. Compared with the gene sequences of a large number of sheep pox virus, goat pox virus, and bovine nodular skin disease virus included in GenBank, it is found that the open reading frame of the virus genus sequence The ORF130 gene sequence is highly conservative. The conserved region in the ORF130 gene sequence is determined by sequence alignment, and primers and probes are designed for this region to detect viruses in the goat pox virus genus. After a lot of design and screening, CaPV-130-F2 and CaPV-130-R2 are the upstream and downstream primers, and CaPV-130-P2 is the probe. The nucleotide sequence of the primer set and probe is:

[0047]CaPV-130-F2: 5'-TGGAAGCAAGTGRTAACGK-3' (SEQ ID NO:1);

[0048]CaPV-130-R2: 5'-CTATTCTATCCCATCATTATACGTA-3' (SEQ ID NO: 2);

[0049]CaPV-130-P2: 5'-TCATCAAAAGGAAACGGATC-3' (SEQ ID NO: 3).

[0050]The fluorescent group labeled at the 5'end of CaPV-130-P2 is FAM, and the quenching group labeled at the 3'end of the probe sequence is MGB.

[0051]The primers and probes of this group are designed for bovine nodular skin disease virus. Genetic analysis software is used to compare the sequence differences between goatpox vaccine strain CVCC AV41 and bovine nodular skin disease virus. It was found that on the ORF132 gene fragment, compared to the gene sequence of the bovine nodular skin disease virus, the goat pox vaccine strain lacked 18 bases, and the position of the deleted bases was from the 210th nucleotide to the 227th nucleotide. According to the comparison results, the position of the base deletion of the vaccine strain was selected as the target gene region for the differential diagnosis of LSD, and the probe sequence for the bovine nodular skin disease virus was designed. Designed and screened out LSD-132-F5 and LSD-132-R6 as upstream and downstream primers, LSD-132-LP5 as probe, and the nucleotide sequence of the primer set and probe is:

[0052]LSD-132-F5: 5'-TTATCAGATGATTGYGTA-3' (SEQ ID NO: 4);

[0053]LSD-132-R6: 5'-TATAAGAATTGTCGAGAGA-3' (SEQ ID NO: 5);

[0054]LSD-132-LP5: 5'-AGTTCAGTTTTAACATCA-3' (SEQ ID NO: 6).

[0055]The fluorescent group labeled at the 5'end of LSD-132-LP5 is VIC, and the quenching group labeled at the 3'end of the probe sequence is MGB.

[0056]Example 2

[0057](1) Synthesize the target gene sequence of the ovine poxvirus virus (Genbank accession number: KX576657) and the bovine nodular skin disease virus target gene sequence (Genbank accession number: MN072619), link the genes at both ends to the vector, and combine The vector DNA is used as a positive plasmid for PCR amplification.

[0058](2) Using the prepared positive plasmid, use the primer set of Example 1 CaPV-130-F2, CaPV-130-R2 and LSD-132-F5, LSD-132-R6, probe CaPV-130-P2 and LSD-132 -LP5 performs fluorescent PCR amplification on the diluted plasmid standard under the optimal amplification conditions. The amplification system is:

[0059]

[0060]

[0061]The amplification procedure is: 50℃, 3min; 95℃ pre-denaturation 5min; 95℃ denaturation 15s, 58℃ annealing extension 30s, a total of 40 cycles, collect fluorescence signal, select goat pox virus genus, select FAM channel and bovine nodular skin disease Select the VIC channel to obtain the amplification curve.

[0062](3) Results determination: Analyze the amplification curve to determine whether there is a goat pox virus in the sample, and determine whether the virus in the sample is a bovine nodular skin disease virus.

[0063]The result judgment method is:

[0064]①Judgment of test validity

[0065]The positive control has a typical amplification curve in the FAM and VIC channels and the Ct value is ≤30, and the negative control has no Ct value or amplification curve in the FAM and VIC channels, the test result is judged to be valid; otherwise, the test is considered invalid.

[0066]②Sample judgment

[0067]1) Only the FAM channel of the sample to be tested has a typical amplification curve and a Ct value ≤ 38, and it is judged to be a goat pox virus (sheep pox virus or goat pox virus or bovine nodular skin disease virus) nucleic acid positive; if 38

[0068]2) A typical amplification curve in the VIC channel of the sample to be tested and a Ct value ≤ 38 is determined to be positive for bovine nodular skin disease virus; if 38

[0069]The sensitivity and specificity of the method of the present invention are further tested below.