2.7 Comparison of LAMP-CRISPR with Taqman®real-time qPCR from clinical samples
The Taqman real-time qPCR detection of the ASFV B646L (p72) gene was performed using a QuantStudio 5 system according to the procedure recommended from OIE described previously (King et al., 2003). The primers and Taqman probe were shown in Table 1. Briefly, 10 μl of probe Master Mix (Takara, Dalian), 0.4 μl of primer F, 0.4 μl of primer R, 3 μl of DNA and ddH2O. Reactions were conducted in a 25 μl volume following the kit instructions. Reaction cycle parameters were set as denaturation at 95 °C for 5 min, followed by 45 cycles of amplification, 95 °C for 15 s and 58 °C for 60 s. Pigs were infected with 10 HAD50 ASFV (CN/GS/2018). Nasal and blood samples were collected at 1, 3, 5, 7, 9,11,13 and 15 dpi, tissue samples were also collected when pigs were dead. A total of 41 ASFV clinical samples were used to assess the performance of the LAMP-CRISPR. These materials comprised the nasal swab, spleen, liver, lung, submandibular lymph node and kidney samples from pigs collected in Lanzhou Veterinary Research Institute. Meanwhile, the real-time qPCR was also performed in parallel with DNA extracted from those samples.