CRISPR/Cas12a-FBDA for Rapid Visual Diagnostics

The CRISPR/Cas12a-FBDA performed as described previously with minor modifications.[23 (link),24 (link)] The CRISPR/Cas12a reaction was composed of 30 nM of crRNA, 330 nM of EnGen Lba Cas12a (Cpf1) (New England Biolabs, USA), 600 nM of fluorescent probe (5′-FAM-TTATTATT-BHQ1-3′), 1X of NEBuffer 2.0 (New England Biolabs, USA), and 1 μL of RPA amplicons in a total reaction volume of 15 μL. The CRISPR/Cas12a reaction was incubated at 39°C for 20 min. The fluorescent signal was then observed by naked eye using a BluePAD Dual LED Blue/White Light Transilluminator (BIO-HELIX, Taiwan) at 470 nm wavelength. Each test was observed by three certified laboratory technicians who were instructed to identify the qualitative test outcome as positive or negative. The tests were considered positive if at least two of the three technicians read the results as positive. Both investigators and the technicians were unaware of the outcome.