An ultrasensitive and contamination-free on-site nucleic acid detection platform for Listeria monocytogenes based on the CRISPR-Cas12a system combined with recombinase polymerase amplification

Abstract

A simple, precise, and highly sensitive detection method is urgently needed for diagnosis and monitoring of foodborne pathogens. To meet these requirements, we designed a micro-propulsion reactor based on the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA) for simultaneous amplification and detection and named it “Cas12a-MPR”. This method can provide a contamination- and instrument-free nucleic acid rapid detection platform with precision and ultrasensitivity using CRISPR-Cas12a combined with RPA in the same vessel. For improved performance, the interaction between the CRISPR detection system and the RPA reagent was explored. Our data showed that the impact of CRISPR reagents on RPA was negligible, but magnesium ions played a vital role in the functioning of the CRISPR/Cas12a system. After exploring the interaction between the CRISPR detection system and the RPA reagent, the method was further optimized to specifically detect target DNA at the attomolar level and achieved a limit of detection of 10 CFU mL-1 at 37 °C. Compared to qPCR, our detection results exhibited 100 % accuracy for detection of Listeria monocytogenes contamination. All the results demonstrated that Cas12a-MPR has great promise for the detection of foodborne pathogens.