Development of multiplex real-time PCR assays for differential detection
of capripoxvirus, parapoxvirus, and foot-and-mouth disease virus
Abstract
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
-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 differential viruses. 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), 36 (PaPV) and 39 (FMDV) clinical specimens
collected from experimentally (CaPV and FMDV) and naturally (PaPV)
infected animals, and all tested positive (DSe 100%) except two FMDV
specimens that were tested negative (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.