Development of a multiplex TaqMan qPCR assay for simultaneous detection and differentiation of four DNA and RNA viruses from clinical samples of sheep and goats.

Mixed infections with different pathogens are common in sheep and goats under intensive production conditions. Quick and accurate detection and differentiation of different pathogens is necessary for epidemiological surveillance, disease management and import and export controls. Multiplex TaqMan qPCR protocols were developed and subsequently evaluated as effective tools in simultaneously detecting single and mixed infections in sheep and goats. Four pairs of primers and four probes labeled with Rox/BHQ2, Cy5/BHQ2, Hex/BHQ1 and Fam/BHQ1 for peste des petits ruminants virus (PPRV), foot and mouth disease virus (FMDV), goat pox virus (GTPV) and orf virus (ORFV), respectively, were used in the multiplex TaqMan qPCR assay. The assay was shown to be sensitive with detection limits of 9.17 × 101 , 1.69 × 102 , 9.41 × 101 and 7.46 × 101 copies/μL for PPRV, FMDV, GTPV and ORFV from a mixture of four viruses in a reaction, respectively. The assay was highly specific in its ability to detect one or more viruses in various combinations in the specimens. 38 clinical samples collected from sheep and goats were detected among 43 samples tested by multiplex TaqMan qPCR, showing highly effective identification. Overall, the multiplex TaqMan qPCR panel provides a fast, specific, and sensitive diagnostic tool for the accurate detection of multiple viral pathogens in sheep and goats.