Development of multiplex real-time PCR assays for differential detection of capripoxvirus, parapoxvirus, and foot-and-mouth disease virus.

This study reports the development of multiplex real-time PCR assays for differential detection of capripoxvirus (CaPV), parapoxvirus (PaPV), and foot-and-mouth disease virus (FMDV) in sheep, goats and cattle. Three multiplex assays were developed, a capripox (CaP) rule-out assay for simultaneous detection and differentiation of CaPV and PaPV, a FMD rule-out assay for simultaneous detection and differentiation of FMDV and PaPV, and a FMD/CaP rule-out assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays included β-actin gene ACTB as an internal positive control to monitor PCR inhibition and accuracy of nucleic acid extractions. The optimized assays were highly specific to the target viruses (CaPV, PaPV, and FMDV) with no cross-reactivity against other viruses that cause similar clinical signs. Using positive control plasmids as template, the limit of detection (LOD) of the multiplex assays were estimated as 2 CaPV, 7 PaPV, and 15 FMDV copies per assay. The amplification efficiency (AE) and correlation co-efficient (R2 ), estimated from the standard curves (Ct vs. log10 template dilution), were 94-106% and >0.99, respectively, for CaP and FMD rule-out assays, 96-116% (AE) and >0.98 (R2 ), respectively, for CaP/FMD rule-out assays and 91-102% and >0.99, respectively, for the corresponding singleplex assays. The diagnostic sensitivity (DSe) of the multiplex assays was assessed on 35 CaPV and 39 FMDV clinical specimens from experimentally infected (CS-E) animals, and 29 CaPV (LSDV), 28 FMDV and 36 PaPV clinical specimens from naturally infected (CS-N) animals; all tested positive (DSe 100%) except two CS-E FMDV specimens that were tested negative by FMD rule-out and the corresponding singleplex (FMDV) assays (37/39; DSe 95%). The newly developed multiplex assays offer a valuable tool for differential detection of clinically indistinguishable CaPV, PaPV, and FMDV in suspected animals and animals with mixed infections.