Method for detecting integrated copy number of porcine endogenous retrovirus (PERV) through fluorescence quantitative polymerase chain reaction (PCR) and application thereof
A technology of retrovirus and fluorescent quantification, which is applied to the determination/testing of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., which can solve the problems of high cost and poor reaction specificity, and achieve low cost and strong specificity , good repeatability
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Embodiment 1
[0029] Example 1, Fluorescent quantitative PCR detection of integrated copy number of porcine endogenous retrovirus
[0030] 1. Acquisition of PBMC genomic DNA
[0031] EDTA-K derived from miniature pigs 2 Anticoagulated blood (for example, can be collected from the anterior vena cava in the experiment), and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque density gradient centrifugation. The method is as follows: take 2 mL of anticoagulated blood, mix it with RPMI1640 culture medium (purchased from Sciencell, USA) 1:1, carefully add it to the liquid surface of 4 mL of human lymphocyte separation medium, centrifuge at 2000 r / min for 15 min, and use Collect the cells on the interface with a capillary pipette, put them into a centrifuge tube containing 5 mL of RPMI1640 culture solution, mix well, and centrifuge at 1500 r / min for 10 min. Carefully discard the supernatant, add 4mL RPMI1640 culture medium, blow and mix, centrifuge at 1500r / min for 10min,...
Embodiment 2
[0046] Embodiment 2, the preparation of the real-time fluorescent quantitative PCR detection kit of porcine endogenous retrovirus integration copy number
[0047] 9 μl of RealMasterMix and 1 pmol each of the upstream and downstream primers are packaged together to obtain a real-time fluorescent quantitative PCR detection kit for the integrated copy number of porcine endogenous retrovirus. When using, the user can add templates according to the needs of the experiment.
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