AUTHOR=Zhang Yaoguang , Chen Jian , Pan Hao , Ma Xiaojiang , Jiang Li , Zhu Qian , Wu Huanyu , Wang Zhenyu 

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

JOURNAL=Frontiers in Microbiology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2022.888529

DOI=10.3389/fmicb.2022.888529

ISSN=1664-302X

ABSTRACT=<sec><title>Background</title><p>The protozoan parasites including <italic>Entamoeba histolytica</italic>, <italic>Giardia lamblia</italic>, and <italic>Cryptosporidium parvum</italic> 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.</p></sec><sec><title>Methods</title><p>Specific primers and TaqMan probes were designed for the 16S-like SSU rRNA sequence of <italic>E. histolytica</italic>, the gdh sequence of <italic>G. lamblia</italic>, and the 18srRNA sequence of <italic>C. parvum</italic>. 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.</p></sec><sec><title>Results</title><p>The triplex qPCR assay could specifically detect <italic>E. histolytica</italic>, <italic>G. lamblia</italic>, and <italic>C. parvum</italic> 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<sup>2</sup> and 5 × 10<sup>8</sup> copies/μL; qPCR assays were performed with an efficiency of more than 95% and <italic>R</italic><sup>2</sup> 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 <italic>C. parvum</italic> using the triplex qPCR assay.</p></sec><sec><title>Conclusion</title><p>The triplex qPCR established in this study not only provides a rapid, sensitive, specific tool for the simultaneous detection of <italic>E. histolytica</italic>, <italic>G. lamblia</italic>, and <italic>C. parvum</italic>, but also has good practical application value.</p></sec>