4.7 Analysis of mismatch in cDNA samples
To validate the specificity of each crRNA designed for targeting cDNA
samples, a diagnostic CRISPR-Cas12a assay was employed, utilizing
fluorescence signals as indicators. The reaction mixture comprised 50 nM
of Cas12a, 50 nM of crRNA, 1× Cas12a reaction buffer, 120 nM of
single-stranded DNA (ssDNA) reporter, cDNA templates of wild-type, BA.2,
or BA.5 gene sequences, and pure water. Incubation was conducted at 37
°C for 30 minutes, during which real-time fluorescence measurements were
performed with an excitation wavelength of 485 nm and emission
wavelength of 528 nm.