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Background  The protozoan parasites including  Entamoeba histolytica ,  Giardia lamblia , and  Cryptosporidium parvum  can infect the human intestinal tract and cause serious diseases. In this study, we aimed to develop a triplex real-time quantitative PCR (qPCR) for the simultaneous differential detection of these three intestinal protozoa.    Methods  Specific primers and TaqMan probes were designed for the 16S-like SSU rRNA sequence of  E. histolytica , the gdh sequence of  G. lamblia , and the 18srRNA sequence of  C. parvum . A triplex qPCR assay was developed based on single-duplicate experiments to evaluate its limit of detection (LOD), specificity, stability, and reproducibility. Additionally, 163 fecal samples from patients with diarrhea who tested positive for copro-antigen were tested to verify the practicality of the assay.    Results  The triplex qPCR assay could specifically detect  E. histolytica ,  G. lamblia , and  C. parvum  without cross-reactivity amongst the target-specific TaqMan probes of these three intestinal protozoan parasites and did not produce amplification curves for any other non-target species, and had good specificity. Amplification of serial dilutions showed that the triplex qPCR detected as little as 500 copies/μL of standard plasmid DNA. The standard curve displayed good linearity between 5 × 10 2  and 5 × 10 8  copies/μL; qPCR assays were performed with an efficiency of more than 95% and  R  2  values were greater than 0.99. The triplex qPCR assay had good repeatability with intra- and inter-assay coefficients of variation less than 1.92%. Among the 163 fecal samples, four samples were confirmed to be positive for  C. parvum  using the triplex qPCR assay.    Conclusion  The triplex qPCR established in this study not only provides a rapid, sensitive, specific tool for the simultaneous detection of  E. histolytica ,  G. lamblia , and  C. parvum , but also has good practical application value.

frontiers in microbiology

Development and Preliminary Application of a Triplex Real-Time Quantitative PCR Assay for the Simultaneous Detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum