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 × 100 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.
摘要：
呼吸道感染对全球公共卫生构成严重威胁,强调迫切需要快速，准确,和大规模的诊断工具。近年来,CRISPR/Cas（成簇的规则间隔短回文重复/CRISPR相关）系统，结合等温扩增方法，在核酸检测(NAT)中得到了广泛的应用。然而,由于重组酶聚合酶扩增（RPA）和CRISPR/Cas试剂之间的竞争作用，实现包含所有必需组分的单管反应系统具有挑战性。此外，为了实现精准医学，区分细菌和病毒感染至关重要。这里,我们开发了一种新的NAT方法，称为一罐RPA-CRISPR/Cas12a，结合了RPA和CRISPR分子诊断技术，能够同时检测12种常见呼吸道病原体，包括六种细菌和六种病毒。RPA和CRISPR/Cas12a反应用石蜡分离，提供用于RPA反应的独立平台以在与CRISPR/Cas12a系统混合之前产生足够的目标产物。结果可以在LED蓝光下目视观察。一锅RPA-CRISPR/Cas12a方法的灵敏度为2.5×100拷贝/μL质粒,与其他细菌或病毒没有交叉反应。此外,通过测试细菌和病毒咽拭子样本的临床分离株来评估临床实用性，表现良好。因此，我们的一锅-RPA-CRISPR/Cas12a方法显示出在即时检测中准确和大规模检测12种常见呼吸道病原体的巨大潜力.
