Development and validation of a duplex quantitative real-time RT-PCR assay for simultaneous detection and quantitation of foot-and-mouth disease viral positive-stranded RNAs and negative-stranded RNAs.

A simplified, cost-effective, two-step duplex quantitative real-time RT-PCR assay was shown to detect and quantify foot-and-mouth disease virus positive-stranded RNAs and negative-stranded RNAs simultaneously for improved investigation of the state of virus infection and replication. The primers and Taqman probes were selected from the coding regions of 2B gene and 3D gene respectively, which have the least variations among serotypes. Cells infected acutely, tissue samples and single cell samples were used for evaluation of the assay. At the early stages of virus infection in vitro, the replication level reached a peak at 9h.p.i. and the negative strands were detectable until 3h.p.i. The kinetics of ratios of positive strands to negative strands (+RNA/-RNA) in vivo in the liver, kidney and spleen were similar, which demonstrated that the replication dynamics were similar in the three organs. 55 single cell samples out of 187 were positive by both positive strands qPCR and negative strands qPCR, the ratios (+RNA/-RNA) ranged from 15.6 to 1463.4 which showed considerable difference among single cell samples, indicating that active viral replication differs greatly in single cells. A duplex quantitative real-time RT-PCR was validated as effective and reliable.