Multiplex PCR for simultaneous detection and differentiation of sheeppox, goatpox and orf viruses from clinical samples of sheep and goats

Abstract

A multiplex polymerase chain reaction (mPCR) was developed and evaluated for detection of pox viral infections simultaneously using clinical samples from sheep and goats. Specific primers for three pox viruses of sheep and goats including sheeppox virus (SPPV), goatpox virus (GTPV) and orf virus (ORFV) were designed targeting conserved sequences of the DNA binding phosphoprotein (I3L) coding gene of Capripoxvirus (CaPV) and the DNA polymerase (E9L) gene of parapoxvirus for identification of these viruses. The mPCR assay was found to be sensitive for detecting as low as 350pg of viral genomic DNA or 102 copies of standard plasmid of individual targets; and 103 copies of plasmid in a mixture of two or three viruses. The assay was specific for detecting one or more of the viruses in various combinations from clinical specimens. Two hundred and thirty five (n=235) clinical samples from sheep and goats received from different geographical regions of the country for diagnosis of pox infection were evaluated by developed uniplex and mPCR assays. The assay had improved diagnostic sensitivity and specificity over to in-use laboratory diagnostic methods and can be useful for clinical differential diagnosis of these infections in sheep and goats.