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.