Abstract
Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus group A (PoRVA) are recognized as major enteric viral pathogens responsible for porcine viral diarrhea. These viruses exhibit similar clinical manifestations, including vomiting, diarrhea, and dehydration, which complicate differential diagnosis. Therefore, there is an urgent need for a highly sensitive and specific diagnostic method to differentiate these pathogens. In this study, we developed a TaqMan-based one-step quadruplex reverse transcription real-time PCR (RT-qPCR) assay for the simultaneous and differential detection of these four porcine diarrhea viruses. The standard curves demonstrated correlation coefficients (R 2) exceeded 0.990 across a dynamic range of 107.5 - 102.5 TCID50/mL, and amplification efficiency ranged from 90% to 110%. The limit of detection (LOD) were 101.5 TCID50/mL. Specificity analysis showed no cross-reactivity with other related pathogens. The assay exhibited good repeatability, with coefficients of variation (CVs) ranging from 0.15% to 1.41% for intra-assay and 0.09% to 2.09% for inter-assay, respectively. Finally, this method was evaluated for its practicality in the field using 348 clinical fecal samples. The positive rates of PEDV, TGEV, PDCoV, and PoRVA were 43.68%, 0.57%, 26.44%, and 33.91%, respectively. Furthermore, the coinfection rates of PEDV/PDCoV, PEDV/PoRVA, PDCoV/PoRVA, and PEDV/PDCoV/PoRVA were14.37%, 13.51%, 2.01%, and 5.75%, respectively. Compared to singleplex RT-qPCR assays, the quadruplex method demonstrated agreement rates ranging from 99.41% to 100% in detecting these four viral pathogens. In conclusion, the developed quadruplex RT-qPCR assay offers a reliable, sensitive, and accurate tool for the identification of four causative agents of porcine viral diarrhea, making it suitable for clinical diagnosis, disease surveillance, and epidemiological studies.