Primers, probe and kit for detecting type-II porcine lymphotropic herpesvirus and their application
A herpes virus and kit technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve problems such as inability to deduce detection methods
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Embodiment 1
[0046] Example 1 Design and synthesis of primers and probes
[0047] Download all homologous gene sequences of type Ⅱ porcine lymphophilis virus from GenBank. According to all the homologous gene sequences of type Ⅱ porcine lymphophilis virus standard strain (GeneBankNo.AY170317), use DNAStar software for homology comparison, Primer Express5.0 software was used to design primers and TaqMan probes, which were synthesized by Shanghai Shenggong Company. The length of the amplified target fragment is 234bp.
[0048] The nucleotide sequence of the upstream primer is shown in PLHV-2 D F1;
[0049] The nucleotide sequence of the downstream primer is shown in PLHV-2 D R1;
[0050] The nucleotide sequence of the probe used with the above primers is shown in PLHV-2 D P1. The 5'end of the probe is labeled with reporter FAM fluorescent dye, and the other 3'end is labeled with quencher BHQ1 fluorescent dye.
Embodiment 2
[0051] Example 2 The extraction steps of total DNA are as follows:
[0052] Add an equal amount of anticoagulant buffer solution or use an anticoagulant blood collection tube to collect 3~5Ml pig blood samples, mix well, and store at 2~8℃ for 3 days. Centrifuge the blood sample at 5000r / min for 5min, remove the supernatant, suck up the white blood cells in the upper layer of the red blood cells, and transfer them to a non-polluting or non-biologically toxic centrifuge tube for later use. Follow the instructions of Beijing Tiangen Biotechnology Company Viral Genomic DNA / RNA Extraction Kit (DP315) to extract viral DNA (the DNA extraction in this experiment is only based on the DP315 kit, and other commercial viral DNA extraction kits are applicable), use directly or Store in the refrigerator at -20℃ for later use.
Embodiment 3
[0053] Example 3 Establishment of real-time fluorescent quantitative PCR amplification method
[0054] 1. Real-time fluorescent quantitative PCR reaction system
[0055] Using total DNA as the template, perform real-time fluorescent quantitative PCR reaction, that is, in a 20μL reaction system: 10× fluorescent quantitative PCR buffer 2μL, 0.5μL dNTPs (10mmol / L), 0.5μL primer PLHV-2 D F1 (10umol / L), 0.5μL primer PLHV-2 D R1 (10umol / L), 1.0μL magnesium ion (50mmol / L), 0.5μL fluorescent probe (10umol / L), 2μL DNA template, 0.2μL Taq enzyme (5U), And make up to 20μL with DEPC treated water.
[0056] 2. Real-time fluorescent quantitative PCR reaction conditions
[0057] After putting the sample tube into ABI 7500 fluorescent PCR machine, set the following conditions to proceed: 95°C, 3min; 95°C, 10s; 60°C, 15s; 72°C, 40s for a total of 40 cycles. Collect data at the end of each cycle. After the reaction is over, the result is judged according to the amplification curve and the standard ...
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