Development of Multiplex Real-Time Quantitative PCR for the Detection of Giardia duodenalis, Enterocytozoon bieneusi, and Cryptosporidium spp. in Dairy Goats

<i>Giardia duodenalis</i>, <i>Enterocytozoon bieneusi</i>, and <i>Cryptosporidium</i> spp. are common zoonotic intestinal protozoa in dairy goats, capable of cross-species transmission. These pathogens impair the growth and development of dairy goats and present a significant threat to public health. Conventional detection methods are time-consuming and lack sensitivity, requiring a rapid and efficient technique for simultaneous detection. A <i>Taq</i>Man probe-based multiplex real-time qPCR assay for the detection of the three protozoa was established in this study using specific primers and probes. Specificity validation demonstrated no cross-reactivity with other common intestinal pathogens of dairy goats. Sensitivity testing revealed minimum detection limits of 29.83, 39.33, and 33.15 copies/μL for the three protozoa, with excellent repeatability. Detection of clinical fecal samples and comparison with PCR confirmed the assay's superior performance, which was capable of identifying positive samples missed by PCR. In conclusion, this rapid, specific, and sensitive assay provides technical support for large-scale epidemiological surveys and real-time monitoring of related protozoan diseases in dairy goats, thereby laying a scientific foundation for prevention and control and safeguarding both the dairy goat industry and public health.