Rodent pinworms, Spironucleus muris and Tritrichomonas muris are the endoparasites that should be monitored and excluded from laboratory animal colonies. Nevertheless, traditional diagnostic methods may not efficiently detect and accurately demonstrate the endoparasite infestation status. In this study, we developed a multiplex PCR assay targeting the rRNA genes to simultaneously detect and differentiate five endoparasites, including Syphacia obvelata, Syphacia muris, Aspiculuris tetraptera, Spironucleus muris, and T. muris, as well as a housekeeping gene in feces. The multiplex PCR could identify an equivalent infection of pinworm, Spironucleus muris and T. muris, with a detection limit of as few as 10 copies. Furthermore, dual infections with up to 100-fold differences and triple infections with 10-fold differences in parasite loads can also be detected. In comparison of traditional methods with the multiplex PCR assay, 76 rodents from 11 research colonies and 3 pet shops and additional 27 fecal samples from laboratory rodents were screened for the infestation status of the five endoparasites. The multiplex PCR had higher sensitivity (97.2–100%) and accuracy (99–100%) than those of the traditional antemortem (sensitivity: 83–100%; accuracy: 94–100%) and postmortem methods (sensitivity: 75–100%; accuracy: 92.1–100%). In addition, an early stage of S. obvelata contamination in a SPF laboratory animal colony was also successfully detected by this multiplex PCR assay. This Pinworm/Spironucleus/Tritrichomonas/Actin Multiplex PCR assay should be a powerful tool to screen endoparasite infestations in laboratory colonies without animal sacrifice.
