A triplex real-time PCR for differential detection of classical, variant and Bartha-K61 vaccine strains of pseudorabies virus
Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous detection and differentiation of three Office International des Epizooties (OIE) classified vesicular viruses: foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease, causing clinically indistinguishable vesicular diseases in swine. The multiplex assay consists of extraction of total RNA from clinical samples; reverse transcription to cDNA using random primers and one-tube real-time amplification of cDNA using multiplex PriProET with specific fluorescent-labelled primers and probes for detection of the three viruses from the vesicular disease complex. The probes are labelled with unique reporter fluorophores, which during amplification are excited by donor fluorophores incorporated in the 5' end of specific amplicons by primer extension. The sensitivity of the multiplex assay was approximately 100 TCID(50), which is 10-fold lower compared to the individual PriProET assays for the three vesicular viruses.