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

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.