Rapid, sensitive, and specific point-of-care testing for pathogens is crucial for disease control. Lateral flow assays (LFAs) have been employed for nucleic acid detection, but they have limited sensitivity and specificity. Here, we used a fusion of catalytically inactive SpCas9 endonuclease and VirD2 relaxase for sensitive, specific nucleic acid detection by LFA. In this assay, the target nucleic acid is amplified with biotinylated oligos. VirD2-dCas9 specifically binds the target sequence via dCas9 and covalently binds to a FAM-tagged oligonucleotide via VirD2. The biotin label and FAM tag are detected by a commercially available LFA. We coupled this system, named Vigilant ( Vi rD2-dCas9 g u i ded and L FA-coupled n ucleic acid t est), to reverse transcription-recombinase polymerase amplification to detect SARS-CoV2 in clinical samples. Vigilant exhibited a limit of detection of 2.5 copies/μL, comparable to CRISPR-based systems, and showed no cross-reactivity with SARS-CoV1 or MERS. Vigilant offers an easy-to-use, rapid, cost-effective, and robust detection platform for SARS-CoV2.
【저자키워드】 COVID-19, SARS-CoV-2, biosensors, Nucleic acid detection, RT-RPA, lateral flow assay, CRISPR-Cas9, VirD2, dCas9, molecular diagnostics, relaxases, 【초록키워드】 SARS-CoV2, MERS, cross-reactivity, CRISPR, point-of-care, amplification, clinical samples, nucleic acid, pathogen, Nucleic acid detection, Sensitivity and specificity, disease control, limit of detection, Rapid, Pathogens, dCas9, fusion, disease, recombinase polymerase amplification, platform, Biotin, this system, sequence, polymerase, offer, robust, bind, amplified, detect, exhibited, comparable, FAM, inactive, biotinylated, catalytically, Lateral, 【제목키워드】 SARS-CoV2, detection, Flow, Lateral,