A double pcr detection primer, kit and detection method for differentiating sheep oral disease virus and sheep pox virus
A sheep aphthous virus and sheep pox virus technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc. Quantitative, sensitive and specific effects
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Embodiment 1
[0032] Example 1 Primer Design
[0033] According to the genome sequences of sheep pox virus, goat pox virus, sheep pox virus and bovine pimple skin disease virus in GenBank, 2 pairs of primers were designed, among which the ORFV primer pair can cover more sheep pox virus to the greatest extent; the CaPV primer pair can amplify Goat pox virus, sheep pox virus and bovine pimple skin disease virus. Its base sequence is shown below.
[0034] ORFV-F: CATCATTGCCGTTTACCTCAGA (SEQ ID NO: 1) or CATCATTGCCGTTTACTTCAGA (SEQ ID NO: 2);
[0035] ORFV-R: TAAGATCYGTGTCGTGTATTCC (SEQ ID NO: 3) or TAAGTTCYGTGTCGTGTATTCC (SEQ ID NO: 4);
[0036] CaPV-F: CTGGAAGCAAGTGGTAACGGAATA (SEQ ID NO: 5);
[0037] CaPV-R: AACGATACGAAGGAAATGTGGG (SEQ ID NO: 6).
Embodiment 2
[0038] Example 2 Double PCR detection method
[0039] 1) Extraction of DNA
[0040] Viral nucleic acid was extracted according to the instructions of Axygen's DNA / RNA extraction kit.
[0041] 2) Double PCR amplification reaction
[0042] A double-PCR detection method was established by using the nucleic acid mixture of sheep aphthus virus and (or) sheeppox virus as the template of double-PCR. Use 50 μL PCR reaction system: 25 μL 2×Taq PCR Master Mix, 1 μL each of primers ORFV-F, ORFV-R, CaPV-F and CaPV-R, 5 μL of ORFV and CaPV templates, sterilized H 2 O 11 μL.
[0043] PCR reaction program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 30 s, a total of 40 cycles; final extension at 72°C for 10 min.
[0044] Observation of results: Take 30 μL of PCR products for 1.5% agarose gel electrophoresis analysis, and observe the results under the gel imaging system.
Embodiment 3
[0045] Embodiment 3 specificity test
[0046] According to the method of embodiment 2, utilize double PCR detection method to positive recombinant plasmid and Peste des petits ruminants virus, bovine viral diarrhea and mucosal disease virus, blue tongue disease virus, fowlpox virus, foot-and-mouth disease virus by double PCR detection method to test.
[0047] see results figure 1 . Lane M: DNA Marker 2000; 1: ORFV positive plasmid; 2: CaPV positive plasmid; 3: ORFV+CaPV positive plasmid; 4: bovine viral diarrhea and mucosal disease virus nucleic acid; 5: bluetongue virus nucleic acid; 6: small Nucleic acid of Peste ruminant disease virus; 7: Nucleic acid of fowlpox virus; 8: Nucleic acid of foot-and-mouth disease virus.
[0048] Depend on figure 1 It can be seen that the optimized double PCR detection method was used to amplify the positive recombinant plasmids and mixtures of oral ulcer virus and sheep pox virus, and the results obtained fragments of about 600bp and 300bp...
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