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).