New duplex qPCR test distinguishes wild-type from persistent O foot-and-mouth virus

Foot-and-mouth disease, caused predominantly by serotype O foot-and-mouth disease virus (FMDV), remains one of the most contagious and economically damaging animal diseases worldwide. A persistent obstacle to controlling it is that recovered or vaccinated animals can become asymptomatic carriers of persistent strains, known as Op strains, which current diagnostic methods cannot distinguish from acute wild-type (WT) strains.

Researchers now report the development of a duplex TaqMan real-time PCR assay designed to detect and discriminate between wild-type and persistent O FMDV strains in a single reaction. The work, published in Applied Microbiology and Biotechnology as an open-access research article, is based on comparative next-generation sequencing and genomic analysis of WT and persistent O FMDV strains.

The assay design exploited two distinct genomic features. Conserved regions of the viral genome were identified to serve as targets for universal detection, allowing the co-detection channel to pick up both wild-type and persistent strains. In parallel, the team targeted single nucleotide polymorphism (SNP) mutations that are specific to persistent Op strains, using amplification refractory mutation system PCR (ARMS-PCR) to achieve differential amplification in a separate typing channel.

The optimized assay therefore consists of a co-detection channel and a typing channel operating together. According to the study, the assay demonstrated excellent specificity, with no cross-reactivity against host nucleic acids or unrelated cell lines, and it achieved a detection limit of 5 copies per microliter. The typing channel produced clear differentiation of WT from Op strains.

In laboratory testing, the duplex assay successfully distinguished wild-type and persistent strains in infected cell samples and across different viral passages, specifically at passages 30 and 60 (PI30 and PI60). This ability to follow Op strains through successive passages underpins the assay’s stated purpose of monitoring viral persistence in experimental systems.

The authors describe the assay as the first duplex TaqMan RT-qPCR test to differentiate wild-type and persistent O FMDV strains, and they position it as a sensitive, specific, and efficient tool for simultaneous detection and differentiation in infected cell samples and across viral passages. The key points accompanying the article note that the assay differentiates Op strains at passages 30 and 60 and that it may aid disease control by enabling persistence monitoring.

Important limitations accompany these findings. The authors state explicitly that clinical validation remains to be performed, meaning the assay has not yet been evaluated on clinical samples from animals in the field. The reported performance figures, including the detection limit and the absence of cross-reactivity, were established in the laboratory context of infected cell samples and viral passages rather than in diagnostic settings involving live animals.

Despite that limitation, the study’s authors present the assay as a promising basis for further research on FMD surveillance and the detection of viral persistence. If validated clinically, a test that can tell whether FMDV detected in a sample comes from an acute wild-type infection or from a persistent carrier state could give disease-control programs a new instrument for understanding and managing the carrier problem that has long complicated foot-and-mouth disease eradication efforts.

Subject of Research: Development of a duplex qPCR assay for detecting and differentiating wild-type and persistent O foot-and-mouth disease virus

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

Article References: Han, L., Du, Y., Liu, F., Chen, X., & Yuan, Y. (2026). Duplex qPCR for detection and discrimination of wild-type and persistent O foot-and-mouth disease virus. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14080-w

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14080-w

Keywords: foot-and-mouth disease virus, duplex qPCR, TaqMan RT-qPCR, persistent infection, carrier animals, single nucleotide polymorphism, ARMS-PCR, viral surveillance, serotype O, diagnostic assay, next-generation sequencing, detection limit