OPERA-Cas12a: A streamlined one-pot system for specific and sensitive on-site detection of nucleic acids

Rapid, sensitive, and user-friendly methods are essential for the detection of nucleic acid. The combination of isothermal amplification with CRISPR/Cas12a not only demonstrates significant advantages but also holds promising prospects for widespread application. However, the requirement for separate nucleic acid preamplification and Cas12a cleavage complicates operational procedures and increases the risk of aerosol pollution. Developing an easily implementable and widely applicable one-pot system remains a challenge. In this study, we introduced a novel approach to balance the reaction kinetics between enzymatic recombinase amplification (ERA) and Cas12a enzyme activity to establish a highly sensitive one-pot ERA/Cas12a (OPERA-Cas12a) system. We systematically identified key factors affecting reactions and utilized a definitive screening design (DSD) optimization strategy to streamline the optimization process. Using porcine-specific gene as the detection target, we successfully established the OPERA-Cas12apork system for pork detection, which demonstrated high specificity and sensitivity. Extending this strategy to duck-specific gene, we developed the OPERA-Cas12aduck system with comparable performance. Both OPERA-Cas12a systems achieved a limit of detection (LOD) of 6 copies per reaction within 60 minutes. Moreover, the detection results can be conveniently read with an LED blue light illuminator, and images can be captured using a smartphone. This OPERA-Cas12a system offers a valuable reference for developing other CRISPR/Cas-base nucleic acids detection applications.