A Multiplex PCR Assay for Simultaneous Detection of Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp. and Enterocytozoon bieneusi in Goats

<i>Giardia duodenalis</i>, <i>Cryptosporidium parvum</i>, <i>Blastocystis</i> spp. and <i>Enterocytozoon bieneusi</i> are four common zoonotic parasites associated with severe diarrhea and enteric diseases. In this study, we developed a multiplex PCR assay for the simultaneous detection of these four zoonotic protozoans in goat stool samples and assessed its detection efficiency. Specific primers were designed from conserved gene sequences retrieved from GenBank, and the PCR conditions were optimized. Genomic DNA from 130 samples was subjected to both single-target PCR and multiplex PCR. The multiplex PCR assay successfully amplified specific gene fragments (<i>G. duodenalis</i>, 1400 bp; <i>C. parvum</i>, 755 bp; <i>Blastocystis</i> spp., 573 bp; <i>E. bieneusi</i>, 314 bp). The assay sensitivity was ≥10² copies of pathogenic DNA clones with high specificity confirmed by negative results for other intestinal parasites. The detection rates were 23.08% (30/130) for <i>G. duodenalis</i>, 24.62% (32/130) for <i>C. parvum</i>, 41.54% (54/130) for <i>Blastocystis</i> spp., and 12.31% (16/130) for <i>E. bieneusi</i>, matching the single-target PCR results. The sensitivity and predictive values were 100.00%. This multiplex PCR provided a rapid, sensitive, specific, and cost-effective approach for detecting these four parasites. It also provided essential technical support for the rapid detection and epidemiological investigation of <i>G. duodenalis</i>, <i>C. parvum</i>, <i>Blastocystis</i> spp., and <i>E. bieneusi</i> infections in goat fecal samples.