Simultaneous Dual‐Gene Diagnosis of SARS‐CoV‐2 Based on CRISPR/Cas9‐Mediated Lateral Flow Assay

Few methods for the detection of SARS‐CoV‐2 currently have 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. Developed here is 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 samples showed 100 % negative predictive agreement and 97.14 % positive predictive agreement. This platform will provide a more accurate and convenient pathway for diagnosis of COVID‐19 or other infectious diseases in low‐resource regions. A CRISPR/Cas9‐mediated triple‐line lateral flow assay was developed through combination with multiplex reverse transcription‐recombinase polymerase amplification (RT‐RPA) for simultaneous dual‐gene diagnosis of SARS‐CoV‐2. With this platform, the cell‐cultured SARS‐CoV‐2, SARS‐CoV‐2 viral RNA standards, and nasopharyngeal swab clinical samples could be sensitively and specifically detected.