A novel five-plex digital PCR assay for the simultaneous detection of murine pathogens: Sendai virus, reovirus, mouse parvoviruses, pneumonia virus of mice, and mouse hepatitis virus

Animal viruses are significant factors that threaten the safety of laboratory animals and are related to the stability of laboratory animal quality. Early detection and diagnosis are crucial for effectively preventing and controlling viral contamination. Digital PCR technology is a novel detection technique that can greatly increase the sensitivity of detection methods. However, few reports exist on the use of digital PCR for detecting viruses in laboratory animals. Thus, in this study, we focused on five experimental animal viruses—Sendai virus (SeV), reovirus 3 (REO3), mouse parvovirus (MPV), pneumonia virus of mice (PVM), and mouse hepatitis virus (MHV)—that infect laboratory mice and rats relatively easily. A multiplex digital PCR assay using five distinct fluorescence signals (FAM, HEX, ROX, Cy5, and Cy3) was established for the simultaneous detection of viruses in experimental animals. Through a series of experiments, including optimization of the reaction system, specificity testing, sensitivity testing, and repeatability testing, a five-plex digital PCR method was developed. The results showed that five-plex dPCR could specifically detect target virus genes, with no specific amplification of VSV, SVA, MCMV, PPV, or PTV. The multiplex dPCR standard curves exhibited good linear relationships, with R 2 values all above 0.99. Sensitivity experiments were conducted to determine the detection limits of the multiplex dPCR method for various viruses: REO3 at 1.21 × 10 1 copies/μL, MPV at 1.27 × 10 1 copies/μL, MHV at 0.87 × 10° copies/μL, SeV at 1.62 × 10° copies/μL, and PVM at 1.52 × 10 1 copies/μL. Reproducibility experiments indicated that the within-batch and between-batch coefficients of variation for dPCR were 0.55–2.78% and 0–2.86%, respectively. The proposed method was proven to be reproducible. A total of 161 clinical samples were tested to evaluate the effectiveness of the method, with dPCR positivity rates for MHV, SeV, MPV, PVM, and REO3 of 0%, 16.8%, 3.1%, 3.1%, and 0.6%, respectively. Therefore, we successfully constructed a five-plex digital PCR system, laying a solid foundation for the rapid diagnostic detection of pathogens to improve health surveillance programs in laboratory animal facilities. IMPORTANCE Digital PCR technology has been successfully applied to the detection of various viruses. Sendai virus (SeV), reovirus 3 (REO3), mouse parvovirus (MPV), pneumonia virus of mice (PVM), and mouse hepatitis virus (MHV) have seriously affected the health of experimental animals. To detect the five pathogens simultaneously, a multiplex digital PCR (dPCR) was established in this study. Furthermore, the five-plex digital PCR was rigorously evaluated, focusing on the limit of detection (LOD), sensitivity, specificity, and reproducibility. The results demonstrated excellent repeatability and specificity of the assay, which was successfully validated using 161 clinical samples. Our study provided a rapid and sensitive method for the simultaneous detection of five major viral pathogens affecting experimental animals.