Development of One-step Multiplex RT-PCR Method for Simultaneous Detection and Differentiation of Foot-and-mouth Disease Virus Serotypes O, A, and Asia 1 Circulating in Vietnam

A one-step multiplex RT-PCR method using new primers was developed for the simultaneous detection and differentiation of Vietnamese FMDV serotypes O, A, and Asia 1 directly from clinical samples. The RT-PCR method used a cocktail of one universal minus-sense primer designed in the 2B gene and three serotype-specific plus-sense primers designed in the hypervariable regions of the capsid VP1 coding gene of FMDV. These serotype-specific primer pairs amplified 658, 535, and 427 bp PCR products corresponding to FMDV serotypes O, Asia 1, and A, respectively. In this study, six well-characterized FMDV strains belonging to serotypes O, A, and Asia 1 were used as reference strains for validation tests. Among these six FMDV strains were three vaccine strains for type O (O1/Manisa), A (A22/Iraq), and Asia 1 (As1/Shamir/89). The other reference strains included one pandemic strain of FMDV serotype Asia 1 (Asia1/MOG/05) and two pandemic strains of FMDV serotype O (O/UKG/34/2001 and O/SKR/2000). For field application, 37 positive-clinical samples and 18 cell culture-adapted viruses belonging to serotypes O, A, and Asia 1, as confirmed previously by antigen ELISA for FMDV detection, were used. The present method showed high sensitivity and specificity and can be adapted for detection and typing of FMDV serotypes O, A, and Asia 1 circulating in Vietnam.