Development of a single-tube, dual-target CRISPR Cas12a/Cas13a system for rapid screening of coinfection with respiratory syncytial virus and rhinovirus.

Background: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are leading causes of respiratory infections in children, with increasing reports of coinfections leading to severe complications. Current CRISPR-based detection systems, such as Cas12a and Cas13a, are limited in multiplex detection due to the lack of specific reporter cleavage mechanisms. This study aims to develop a rapid, sensitive, and single-tube dual-gene detection method for RSV and HRV using the orthogonal trans-cleavage activities of CRISPR-Cas12a/13a combined with reverse transcription-recombinase polymerase amplification (RT-RPA).

Methods: We designed a novel detection system leveraging RT-RPA for amplification and the distinct cleavage activities of Cas12a and Cas13a for simultaneous dual-gene detection.

Results: The reaction components were optimized to complete detection within 30 min, achieving sensitivities of 10 copies/µL for RSV and 10² copies/µL for HRV. Clinical validation was performed on 543 respiratory infection samples, confirming high accuracy and specificity.

Conclusions: The RT-RPA-CRISPR-Cas12a/13a system provides a rapid, sensitive, and efficient solution for RSV and HRV coinfection detection. This method supports early diagnosis and improved clinical management, offering significant potential for public health applications in preventing severe respiratory complications in children.