RT-RPA-Cas12a-based assay facilitates the discrimination of SARS-CoV-2 variants of concern
Guiyue Tang, Zilong Zhang, Wei Tan, Fei Long, Jingxian Sun, Yingying Li, Siwei Zou, Yujiao Yang, Kezhu Cai, Shenwei Li, Zhiyi Wang, Jiakun Liu, Guobing Mao, Yingxin Ma, Guoping Zhao, Zhengan Tian, Wei Zhao
Abstract
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 ΔH69-V70 were discriminated by RRCd, demonstrating 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.