RT-RPA-Cas12a-based 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. However, current methods are limited by the low sensitivity, long turn-around time or high cost. Here, we report a nucleic acid testing-based method aiming to detect and discriminate SARS-CoV-2 VOCs by combining <underline>R</underline> T- <underline>R</underline> PA and <underline>C</underline> RISPR-Cas12a <underline>d</underline> etecting assays (RRCd). With a detection limit of 10 copies RNA/reaction, RRCd was validated in 204 clinical samples, showing 99% 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 ΔH69-V70 were discriminated by RRCd, demonstrating 100% accuracy in clinical samples with C _t < 33. The method completes within 65 min and could offer visible results without using any electrical devices, which may facilitate point-of-care testing of SARS-CoV-2 and its variants.