CRISPR-Cas12a combined with reverse transcription recombinase polymerase amplification for sensitive and specific detection of human norovirus genotype GII.4

Abstract

Human norovirus (NOV) is a common and serious virus that accounts for sporadic cases and outbreaks of gastroenteritis. This study aimed to develop rapid, reliable and portable detection systems by coupling reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a (RT-RPA-Cas12a) for NOV genotype GII.4. Here, three primers for RNA-dependent RNA polymerase gene of NOV were designed and screened. Then, RT-RPA products were detected using CRISPR-Cas12a system by combing with fluorescence or lateral flow (LF). RT-RPA-Cas12a-based fluorescence or LF assay can be completed within 40 min, with the detection limit of up to 9.65 × 10²copies/mL and no cross-reactivity with metapneumovirus, bocavirus, seoul virus, and respiratory syncytial virus. Furthermore, the detection coincidence rates of RT-RPA-Cas12a-based fluorescence and LF with qRT-PCR were 98.3%. Therefore, the present study suggests that both RT-RPA-Cas12a-based fluorescence and LF are promising sensitive, specific and alternative method for diagnosis of NOV genotype GII.4 without ancillary equipment.

Keywords: CRISPR-Cas12a; Genotype GII.4; Human norovirus; Lateral flow; RT-RPA.