ArticleMicrobiology spectrum2026
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.
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Abstract
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 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.
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