Simple, sensitive, and visual detection of 12 respiratory pathogens with one‐pot‐RPA‐CRISPR/Cas12a assay

Abstract Respiratory infections pose a serious threat to global public health, underscoring the urgent need for rapid, accurate, and large‐scale diagnostic tools. In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated) system, combined with isothermal amplification methods, has seen widespread application in nucleic acid testing (NAT). However, achieving a single‐tube reaction system containing all necessary components is challenging due to the competitive effects between recombinase polymerase amplification (RPA) and CRISPR/Cas reagents. Furthermore, to enable precision medicine, distinguishing between bacterial and viral infections is essential. Here, we have developed a novel NAT method, termed one‐pot‐RPA‐CRISPR/Cas12a, which combines RPA with CRISPR molecular diagnostic technology, enabling simultaneous detection of 12 common respiratory pathogens, including six bacteria and six viruses. RPA and CRISPR/Cas12a reactions are separated by paraffin, providing an independent platform for RPA reactions to generate sufficient target products before being mixed with the CRISPR/Cas12a system. Results can be visually observed under LED blue light. The sensitivity of the one‐pot‐RPA‐CRISPR/Cas12a method is 2.5 × 10 0 copies/μL plasmids, with no cross‐reaction with other bacteria or viruses. Additionally, the clinical utility was evaluated by testing clinical isolates of bacteria and virus throat swab samples, demonstrating favorable performance. Thus, our one‐pot‐RPA‐CRISPR/Cas12a method shows immense potential for accurate and large‐scale detection of 12 common respiratory pathogens in point‐of‐care testing.