Development and application of a TaqMan probe-based pentaplex qPCR for the rapid detection and differential identification of five pathogens.

Cryptosporidium parvum (Cp), Strongyloides stercoralis (Ss), Trypanosoma cruzi (Tc), Theileria ovis (To) and Echinococcus granulosus (Eg) are significant in causing similar clinical symptoms in both humans and animals. These symptoms include diarrhea, fever, pneumonia, hepatitis, encephalitis, cardiovascular, and gastrointestinal diseases, as well as reproductive system diseases. Therefore, it is urgent to establish a rapid, specific, and sensitive method to simultaneously detect these five pathogens. Here, we develop a TaqMan probe-based pentaplex real-time fluorescence quantitative PCR (qPCR) for simultaneous detection of Cp, Ss, Tc, To, and Eg for the first time. Five sets of specific primers and probes were designed. The 5' end of five probes were labeled with five different fluorescent groups (FAM, VIC, ABY, JUN, CY5) that do not interfere with each other, while the 3' end were labeled with quenching groups (MGB-NFQ, QSY). The instrument collects fluorescence signals at different wavelengths in five independent optical channels in qPCR to achieve simultaneous specific amplification and quantitative detection of five targets within one tube. A five-color qPCR was developed, which can rapidly and quantitatively distinguish and identify Cp, Ss, Tc, To, and Eg in a single tube. This method shows strong specificity and no cross-reaction with other control pathogens. This method has high sensitivity, with the LLOQ of 10 copies and the LOD as low as 1 copy. The standard curve shows a good linear relationship. The amplification efficiency is high. The repeatability and reproducibility are excellent. The interference test shows that high concentrations of nucleic acids do not interfere with low concentrations of nucleic acids, and can ensure excellent detection results even in complex samples for 1.5 hours. A total of 246 clinical samples were detected: 4.88% were positive for Cp, 2.44% for Ss, and 15.85% for To, while 0.81% for co-infections of Cp/To. This TaqMan probe-based pentaplex qPCR enables rapid detection and differential identification of five pathogens from clinical samples, eliminating the need for multiple single-plex qPCR. It can potentially assist clinicians in diagnosis and treatment planning by rapidly detecting pathogens in clinical samples and further quantitative analysis. It is of extremely significant importance for protecting human health.