Development of a CRISPR-Cas13a assay for mouse hepatitis virus detection.

Mouse hepatitis virus (MHV) is a significant pathogen that undermines the health of laboratory animals and the reliability of research data. The development of rapid and sensitive detection methods for MHV is therefore crucial for ensuring laboratory animal quality and maintaining laboratory biosafety. Here, we established a CRISPR-Cas13a-based assay targeting the E, M, and N genes of MHV by constructing a recombinase-aided amplification (RAA)-coupled detection system. Results demonstrated that the N1 target exhibited the highest sensitivity, achieving a detection limit of 0.1 copy/μL for plasmid templates and 1 copy/μL in spiked serum samples. The system also exhibited robust specificity and a strong signal response in spiked serum samples. Furthermore, we developed a multiplex amplification-free CRISPR-Cas13a assay by combining Cas13a/crRNAs targeting different genes, which achieved a detection limit of 3.06 pM. In summary, this study presents two distinct CRISPR-Cas13a-based strategies for MHV detection, demonstrating the considerable application potential of this technology for pathogen detection in laboratory animal settings.