Enhanced detection and serotyping of foot‐and‐mouth disease virus serotype O, A, and Asia1 using a novel multiplex real‐time RT‐PCR

Rapid and accurate detection and serotyping of foot‐and‐mouth disease (FMD) virus (FMDV) is essential for implementing control policies against emergent FMD outbreaks. Current serotyping assays, such as VP1 reverse transcription‐polymerase chain reaction (RT‐PCR)/sequencing (VP1 RT‐PCR/sequencing) and antigen detection enzyme‐linked immunosorbent assay (ELISA), have problems with increasing serotyping failure of FMDVs from FMD outbreaks. This study was conducted to develop a multiplex real‐time RT‐PCR for specific detection and differential serotyping of FMDV serotype O, A, and Asia 1 directly from field clinical samples. Primers and probes were designed based on 571 VP1 coding region sequences originated from seven pools. Multiplex real‐time RT‐PCR using these primers and probes demonstrated serotype‐specific detection with enhanced sensitivity compared to VP1 RT‐PCR/sequencing for reference FMDV (n = 24). Complete serotyping conformity between the developed multiplex real‐time RT‐PCR and previous VP1 RT‐PCR/sequencing was demonstrated using FMDV field viruses (n = 113) prepared in cell culture. For FMDV field clinical samples (n = 55), the serotyping rates of multiplex real‐time RT‐PCR and VP1 RT‐PCR/sequencing were 92.7% (51/55) and 72.7% (40/55), respectively. Moreover, the developed multiplex real‐time RT‐PCR demonstrated improved FMDV detection (up to 33.3%) and serotyping (up to 67.7%) capabilities for saliva samples when compared with 3D real‐time RT‐PCR and VP1 RT‐PCR/sequencing, during 10 days of challenge infection with FMDV serotype O, A, and Asia 1. Collectively, this study suggests that the newly developed multiplex real‐time RT‐PCR assay may be useful for the detection and differential serotyping of FMDV serotype O, A, and Asia 1 in the field.