Duplex qPCR for detection and discrimination of wild-type and persistent O foot-and-mouth disease virus

Abstract Foot-and-mouth disease (FMD), predominantly caused by serotype O foot-and-mouth disease virus (FMDV), remains highly contagious and economically devastating worldwide. A significant challenge in disease control is that recovered or vaccinated animals can become asymptomatic carriers of persistent strains (Op), which are indistinguishable from acute wild-type (WT) strains using current diagnostic methods. In this study, we developed a duplex TaqMan real-time PCR assay based on comparative next-generation sequencing and genomic analysis of WT and persistent O FMDV strains. Conserved regions were identified for universal detection, and Op-specific single nucleotide polymorphism (SNP) mutations were targeted for differential amplification using ARMS-PCR (amplification refractory mutation system PCR). The optimized assay, consisting of a co-detection channel and a typing channel, demonstrated excellent specificity with no cross-reactivity against host nucleic acids or unrelated cell lines, a detection limit of 5 copies/µL, and clear differentiation of WT from Op strains. Furthermore, the duplex assay successfully distinguished wild-type and persistent strains in infected cell samples and across different viral passages (PI30 and PI60). This duplex TaqMan RT-qPCR assay provides, for the first time, a sensitive, specific, and efficient tool for simultaneous detection and differentiation of wild-type and persistent O FMDV strains in infected cell samples and across viral passages. Although clinical validation remains to be performed, this assay offers a promising basis for further studies on FMD surveillance and the detection of viral persistence. Key points • First duplex TaqMan RT-qPCR assay differentiating wild-type and persistent O FMDV. • Detection limit of 5 copies/µL with no cross-reactivity against host nucleic acids. • Differentiates Op strains at passages 30 and 60, monitors persistence, aids control.