Simultaneous dual-gene diagnosis of SARS-CoV-2 based on CRISPR/Cas9-mediated lateral flow assay.

Xiong, Erhu; Jiang, Ling; Tian, Tian; Hu, Menglu; Yue, Huahua; Huang, Mengqi; Lin, Wei; Jiang, Yongzhong; Zhu, Debin; Zhou, Xiaoming

Accurate and large-scale screening of infected individuals has proven to be an effective means to control the spread of COVID-19. Currently, many assays have been developed to meet the huge testing requirements and the availability of diverse detection settings. However, few methods emphasize the capability to simultaneously detect two genes in a single test, which is a key measure to improve detection accuracy, as adopted by the gold standard RT-qPCR method. Herein, we developed a CRISPR/Cas9-mediated triple-line lateral flow assay (TL-LFA) combined with multiplex reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and simultaneous dual-gene detection of SARS-CoV-2 in a single strip test. This assay is characterized by the detection of envelope (E) and open reading frame 1ab (ORF1ab) genes from cell-cultured SARS-CoV-2 and SARS-CoV-2 viral RNA standards, showing a sensitivity of 100 RNA copies per reaction (25 μL). Furthermore, dual-gene analysis of 64 nasopharyngeal swab clinical samples showed 100% negative predictive agreement (NPA) and 97.14% positive predictive agreement (PPA). Expectantly, this developed platform will provide a more accurate and convenient pathway for diagnosis of COVID-19 or other infectious diseases in low-resource regions.