Development and Optimization of Multiplex PCR for the Identification of A, O and Asia-1 Serotypes of FMDV in Pakistan

Foot and mouth disease (FMD) is highly contagious viral infection of cloven footed animals caused by Aphthovirus of family picornaviridae. Genome of virus is single stranded positive sense RNA. Experiment was conducted to optimize multiplex PCR (mPCR) for rapid detection of circulating serotypes of FMD virus in Pakistan. The serotype-specific primers were selected from VP1 region of FMDV genome responsible for antigenic diversity of the virus. After RNA extraction cDNA was synthesized followed by PCR reaction with serotype specific primers. In multiplex PCR (mPCR) serotype specific primers amplified products of 386, 232 and 240 base pair (bp) for A, O and Asia1 serotypes of FMDV respectively at 56.5 0 C annealing temperature. Sensitivity of multiplex PCR was tested at different concentrations (1.5 l, 2 l and 3 l) of template DNA. It was found to be highly sensitive at 3 l concentration of template DNA. On 20 suspected FMD clinical samples, mPCR showed that it belongs to Asia1 serotypes. The test was found to be very specific for FMDV and exhibited no cross reactivity with peste de petits ruminants virus (PPRV). Multiplex PCR have shown 100% sensitivity on field samples. The test is sensitive and specific and can be used for serotyping of FMDV.