The detection of ASFV by probe-based qPCR has been described previously [29 (link)–31 (link)]. In terms of African swine fever virus (ASFV), qPCR is the gold standard, which we used as a reference for our CRISPR-Cas12a assay. LbCas12a (New England Biolabs) was used for this assay. As described in the DETECTR method, our CRISPR-Cas12a assay also uses recombinase polymerase amplification (RPA). For RPA reactions, the TwistAmp Basic kit (TwistDx) was used according to the manufacturer’s instructions (https://www.twistdx.co.uk/en ). Briefly, 50 μl reactions containing 2.5 μl ASFV DNA, 0.48 μM forward and reverse primers, 1× rehydration buffer, 14 mM magnesium acetate and RPA mix were incubated at 39 °C for 20 min. CRISPR-Cas12a detection was performed as described previously with minor modifications. The reaction volume was a total of 50 μl, with 20 nM crRNA, 115 nM single-stranded DNA fluorophore quencher - labeled reporter (Table S1 ), 30 nM LbCas12a, 1 μl RPA amplification products, 2 μl RNase inhibitor (New England Biolabs), and 1 × LbCas12a Buffer (New England Biolabs). The reactions were incubated in a temperature-controlled water bath for 15 min at 37 °C. Fluorescence emission was excited at 485 nm and detected at 535 nm using a fluorescent microplate reader (BioTek), and reactions without target DNA were used to establish the background.
Free full text: Click here
