RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern

Timely and accurate detection of SARS-CoV-2 variants of concern (VOCs) is urgently needed for pandemic surveillance and control. Great efforts have been made from a mass of scientists in increasing the detection sensitivity and operability, and reducing the turn-around time and cost. Here, we report a nucleic acid testing -based method aiming to detect and discriminate SARS-CoV-2 mutations by combining RT-RPA and CRISPR-Cas12a detecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 194 clinical samples, showing 89% positive predictive agreement and 100% negative predictive agreement, respectively. Critically, using specific crRNAs, representatives of single nucleotide polymorphisms and small deletions in SARS-CoV-2 VOCs including N501Y, T478K and Delta H69-V70 were discriminated by RRCd, demon-strating 100% specificity in clinical samples with Ct < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which probably facilitate point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.

Automated summary

Timely and accurate detection of SARS-CoV-2 variants of concern is essential for pandemic surveillance and control. Scientists have developed a nucleic acid testing method, called RRCd, which combines RT-RPA and CRISPR-Cas12a detecting assays to detect and discriminate SARS-CoV-2 mutations. RRCd has high sensitivity, operability, and can provide results within 65 minutes without electrical devices, making it potentially suitable for point-of-care testing of SARS-CoV-2 variants and other epidemic viruses.