3.2 Quantitative direct detection of ASFV nucleic acid with CRISPR Cas12a
We set out to develop a Cas12a-based direct detection assay for Viral DNA that would avoid the need for expensive instrument and enable point-of-care testing (Figure 1). To develop a sensitive and portable fluorescence detection system, 5 crRNAs were selected to optimize Cas12a activation performance. The results were shown in Figure 2A and 2B. According to the fluorescent intensity and the gradation of fluorescence, the crRNA 4 with the best active performance was selected to perform the CRISPR-Cas12a cleavage reporting reaction. Furthermore, the fluorescent intensity of ssDNA-FQ report 1 and ssDNA-FQ report 2 were compared under the 50ng/μl and 5ng/μl of DNA template. The results revealed that ssDNA-FQ reporter 2 had higher efficiency (Supplementary Figure S4). Moreover, 500 nM of ssDNA-FQ reporter 2 was selected and used to perform our assay (Supplementary Figure S5).