A novel TaqMan-based RT-qPCR assay for the detection of PEDV and discrimination of the G2c subtype.

Porcine epidemic diarrhea virus (PEDV) G2c subtype has emerged as an increasingly prevalent variant causing widespread epidemic, high mortality and severe economic losses in the Chinese swine industry. The lack of specific and efficient detection methods hinders its surveillance, early detection and control. In this study, a TaqMan-MGB probe-based duplex quantitative real-time reverse transcription PCR (RT-qPCR) assay was developed for simultaneous detection of pan-PEDV and differentiation of the G2c subtype. Universal primers/probe targeting the conserved N gene of PEDV and G2c-specific primers/probe targeting the unique mutation sites in the S gene were designed. The assay was validated for performance, including specificity, sensitivity, and repeatability, and further evaluated using artificial challenge models and clinical samples. The standard curves exhibited excellent linearity (R2 > 0.999) with amplification efficiencies of 99.1% (Pan-pedv) and 98.4% (G2c). The limits of detection (LOD) were 10 copies/µL for Pan-pedv and 100 copies/µL for G2c subtype. No cross-reactivity was observed with 11 common swine diarrhea-related pathogens, and the G2c-specific channel exclusively recognized the G2c subtype. Intra-assay and inter-assay coefficients of variation (CVs) were < 1.40% and < 0.62%, respectively, confirming the good repeatability. In the artificial challenge model, the assay detected dynamic changes in fecal viral load consistent with the singleplex RT-qPCR. In clinical sample testing (n = 18), 13 PEDV-positive samples were identified, with 6 G2c-positive cases validated by Sanger sequencing. Collectively, this duplex RT-qPCR assay is sensitive, specific, and reliable, providing a valuable tool for rapid diagnosis, epidemiological surveillance, and targeted control of PEDV G2c subtype outbreaks.