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

BackgroundThe 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. MethodsSpecific 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. ResultsThe 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/mu L of standard plasmid DNA. The standard curve displayed good linearity between 5 x 10(2) and 5 x 10(8) copies/mu L; 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. ConclusionThe 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.

Automated summary

In this study, a triplex real-time quantitative PCR assay was developed for the simultaneous differential detection of three protozoan parasites in the human intestinal tract. The assay showed high sensitivity, specificity, and repeatability, and demonstrated good practical application value in fecal samples from patients with diarrhea.