Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain
A technology of African swine fever virus and fluorescent probe, applied in the field of quadruple quantitative fluorescent probe primer combination, can solve the problems of complex immune evasion mechanism, lack of neutralizing antibody, and no treatment measures
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[0097] 1. Preparation of positive standard
[0098]The positive standards of B646L, EP402R and MGF360-14L were used to extract DNA from the positive samples of known ASFV, and the total DNA was extracted according to the DNA virus extraction kit of Tiangen, using a 25 μL system, and the reaction system included: 2×PCRMix 10 μL , 1 μL upstream primer, 1 μL downstream primer, 2 μL DNA, 0.8 μL TRUEscript Enzyme Mix and RNasefree H 2 O (nuclease-free water) 10.2 μL. PCR amplification program: 94°C for 5min; cycle 94°C for 30s, 55°C for 30s, 72°C for 30s, a total of 30 cycles; then extend at 72°C for 10min. After amplification, all products were identified by 1% agarose gel electrophoresis. PCR products identified as positive were purified and recovered with Tiangen’s glue recovery kit, connected to the pEASY-T1 vector and transformed into DH5ɑ competent cells, and positive clones were picked and amplified by shaking the bacteria with LB culture medium. Sent to Shanghai Sangon B...
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