Fig. 6.

Multiplexed OPTIMA-dx detection with TccCas13a and AapCas12b thermostable Cas enzymes. (A) Schematic representation of one-pot multiplexed OPTIMA-dx detection reaction. The unique collateral activity of Cas12 and Cas13 orthologs enables the use of different reporter molecules with different fluorophores (RNA reporter with FAM fluorophore for Cas13 and DNA reporter with HEX fluorophore for Cas12) and multichannel detection. (B) Performance of multiplexed detection of SARS-CoV-2 (at 400 cp/μL) and isolated human RNA (for RNase P detection) as measured by real-time fluorescence. Data are shown as means ± SD (n = 3). (C) Multiplexed detection of SARS-CoV-2 and the human internal control (RNase P) in RNA extracted from 14 clinical COVID-19 samples. Detection reactions were incubated at 56 °C, and the end-point fluorescence signal was measured with FAM and HEX channels after 1 h. SARS-CoV-2, synthetic SARS-CoV-2 RNA used at 400 cp/μL; RNase P, isolated total human RNA; NTC, no template control. (D) Multiplexed detection of SARS-CoV-2 and the human internal control (RNase P) from 16 clinical oropharyngeal swabs processed with the quick extraction method. Detection reactions were incubated at 56 °C, and the end-point fluorescence signal was measured with FAM and HEX channels after 1 h. -Ve, SARS-CoV-2–negative samples as determined with qRT-PCR. RFU, relative fluorescence unit.