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

## TL;DR

A new rapid and sensitive test was developed to detect three harmful protozoa in dairy goats, improving disease monitoring and public health protection.

## Contribution

A novel TaqMan probe-based multiplex real-time qPCR assay for simultaneous detection of three zoonotic protozoa in dairy goats.

## Key findings

- The assay has detection limits of 29.83, 39.33, and 33.15 copies/μL for Giardia duodenalis, Enterocytozoon bieneusi, and Cryptosporidium spp., respectively.
- The assay outperformed conventional PCR in detecting positive clinical fecal samples.
- The method is specific with no cross-reactivity to other intestinal pathogens in dairy goats.

## Abstract

In this study, a TaqMan probe-based multiplex real-time qPCR assay was established for the simultaneous detection of three common zoonotic intestinal protozoa in dairy goats: Giardia duodenalis, Enterocytozoon bieneusi, and Cryptosporidium spp. The assay was designed using specific primers and probes, and specificity validation confirmed no cross-reactivity with other common intestinal pathogens in 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, compared with PCR, demonstrated the assay’s superior performance, 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, thus laying a scientific foundation for prevention and control and safeguarding both the dairy goat industry and public health.

Giardia duodenalis, Enterocytozoon bieneusi, and Cryptosporidium 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 TaqMan 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.