Visual detection of human metapneumovirus using CRISPR‐Cas12a diagnostics

Abstract

Human metapneumovirus (HmPV) is a common and serious virus that causes respiratory tract infection. This study aimed to develop a detection technique by combining reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for clinical diagnosis of HmPV. Herein, four primer pairs targeting partial nucleoprotein (N) gene of HmPV were designed and evaluated. Then, the products amplified by RT-RPA were detected using CRISPR-Cas12a combined with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 35 min or 45 min, and the detection limit was up to 6.97 × 102 copies/mL. And there was no cross reaction with human bocavirus, respiratory syncytial virus, adenovirus and parainfluenza virus. By combining with LF, the detection results were evaluated by naked eyes. Furthermore, 28 clinical samples were applied to examine the performance of RT-RPA-Cas12a system. The detection coincidence rates of RT-RPA-Cas12a-fluorescence and RT-RPA-Cas12a-LF with quantitative RT-PCR were 96.4% and 92.9%, respectively. Together, the new method for detecting HmPV with high sensitivity and specificity based on RT-RPA-Cas12a-fluorescence or LF shows promising potential for clinical diagnosis of HmPV without professional skills or ancillary equipment.