Ssdna reporter

An LFD assay was established to improve the RPA–CRISPR–Cas12a assay, thereby making it easier to use the assay and interpret the results. The FAM–biotin ssDNA reporter and LFD were designed to capture labeled nucleic acids. The 20 μL RPA–CRISPR–Cas12a–LFD reaction mixture, containing 0.5 μL of LbCas12a nuclease (10 µM), 2 µL of HOLMES Buffer 1 (10×), 1 µL of ssDNA reporter (Sangon, Shanghai, China), 1.5 μL of crRNA A1 (10 µM), 13 μL of nuclease-free water, and 2 μL of RPA products, was mixed thoroughly and incubated at 37.5 °C for 20 min. Then, 10 μL of nuclease-free water was added to the reaction mixture and mixed thoroughly. The LFD was placed in the mixture for 3–5 min. If both the test and control lines were displayed, the result was considered positive. If only the control line was displayed, the result was considered negative. If the control line was not visible, the result was considered invalid, and the test was repeated using a new strip.

The RPA product (10 µL) was transfered to 40 µL of the CRISPR-Cas12a reaction mixture containing 100 nM crRNA (Sangon Biotech, Shanghai, China), 50 nM Cas12a (NEB, Ipswich, UK) and 250 nM ssDNA reporter (Sangon Biotech, Shanghai, China). Then, the reactions (50 µL in a PCR tube) were incubated in a fluorescence plate reader (Molecular Devices, California, USA) or a Real Time PCR Detection System (Bio-Rad, Watford, UK) for up to 60 min at 37 °C with fluorescent signals collected every 30 s (ssDNA FQ substrates = λex: 485 nm; λem: 535 nm).