One-tube and one-buffer system of RT-PCR amplification of 1D gene of foot-and-mouth disease virus field isolates

Abstract

A method of reverse transcription (RT) and polymerase chain reaction (PCR) amplification of 1D (VP1) gene of foot-and-mouth disease (FMD) virus using one reaction mixture containing both avian myeloblastosis virus (AMV) reverse transcriptase (RTase) and Tfl DNA polymerase is described. The procedure was time saving, made use of a single buffer for both RT and subsequent amplification and performed better than the two-step procedure usually conducted with Moloney murine leukemia virus (MMLV) RTase and Taq DNA polymerase for amplification of the VP1 gene of field isolates of FMD virus serotypes O,-A, C and Asia 1. The failure to amplify the VP1 gene of many type O and Asia 1 viruses using MMLV RTase-Taq polymerase enzyme system could be overcome by performing RT of the viral genome at a higher temperature (48 degrees C) with AMV RTase which is not possible with MMLV RTase.