Development and Application of TaqMan-Probe-Based Pentaplex Quantitative Real-Time Polymerase Chain Reaction for Simultaneous Detection of Toxoplasma gondii, Neospora caninum, Eimeria stiedai, Giardia lamblia and Trypanosoma evansi

The emergence of coinfection with Toxoplasma gondii (Tg), Neospora caninum (Nc), Eimeria stiedai (Es), Giardia lamblia (Gl), and Trypanosoma evansi (Te) is an important problem that endangers human health, animal quality and public sanitary security. These pathogenic protozoans play important roles in the establishment of similar clinical signs of diseases in human/sheep/pig/rabbits, including diarrhea, hepatitis, encephalitis, and reproductive disorders. Therefore, a rapid and specific diagnostic method to simultaneously detect Tg, Nc, Es, Gl, and Te is urgently required. Here, we developed a TaqMan-probe-based quantitative real-time polymerase chain reaction (qPCR) for rapid detection of Tg, Nc, Es, Gl, and Te for the first time. Specific primers and TaqMan-probes were designed for the conserved regions in the G3PDH region of Tg, NC5 region of NC, ADF region of Es, GDH region of Gl, and COX1 region of Te, and pentaplex qPCR for simultaneous detection of Tg, Nc, Es, Gl and Te was developed. The assay showed a strong specificity for detecting Tg, Nc, Es, Gl and Te, and had no cross-reactivity with other control pathogenic parasites. The assay had high sensitivity with the lower limit of quantification (LLOQ) and the limit of detection (LOD) are 10 and 3 copies per reaction. The assay exhibited excellent repeatability and reproducibility of the intra-assay coefficient of variation (CV) of 0.07–2.10% and the inter-assay CV of 0.22–2.13%. Finally, the developed TaqMan-probe-based pentaplex qPCR was used to detect 200 clinical samples. The results indicated that the positive detection rate of Tg, Nc, Es, Gl were 2.5% (5/200), 2.5% (5/200), 5.5% (11/200), 33% (66/200), respectively, and the result showed good consistency with conventional PCR. The developed TaqMan-probe-based pentaplex qPCR assay has the advantages of high sensitivity and strong specificity, high throughput, convenient and fast, and cost-effectiveness. It is of great significance to safeguard animal quality and human health.