Comparison of isolation and conventional and developed indoor new sybr green real-time PCR assay for diagnosis of sheep/goat pox and contagious ecthyma viruses

ABSTRACT

Outbreaks of pox among animals have been reported from different parts of the world regularly. Differential diagnosis of Sheep/Goat Pox virus [SGPV], and Contagious ecthyma [Orf] virus [ORFV] by host species and clinical signs is not always exact, hence; laboratory confirmation is necessary to establish the cause of the disease and to distinguish these two viruses. During summer 2009, Pox infection among sheep and goat flocks were suspected. Based on clinical examinations sheep/goat Pox, and contagious ecthyma were suspected; samples of the skin biopsy of affected sheep and goats were sent to the Lab for more exact diagnosis. The etiological viruses were fruitfully isolated on the chorio-aliantoic membranes [CAMs] of embryonated chicken eggs indicated by expressing the characteristic pock lesions Poxfiridae family and confirmed to be a SGPV, and ORFV on the basis of conventional polymerase chain reaction assay [PCR] in sample and CAMs harvests. The primer used for diagnosis of SGPV was Capripoxvirus [KS-15 and KS-1.6], while the primer used for ORFV were 045Orf coding for the later transcription factor [VLTF-1]. A Real-Time polymerase chain reaction assay [RT-PCR], based on SYBR green I technology was subsequently developed for DNA qualitative in sample and CAM harvests for SGPV and ORFV based on the same mentioned primers. The assay was specific as only SGPV or ORFV suspected samples and their reference strains reacted and their reference strains reacted with the corresponding primer only. With melting curve analysis; temperature of melting [T[m]] scored by SGPV were relatively identical between 81.24 [degree sign] C [tilde] 81.49 [degree sign] C while; T[m] scored by ORFV were 92.62[degree sign] C [tilde] 93.08 [degree sign] C. The sensitivity of the RT-PCR with each of the 2 primers used for SGPV or ORFV was equivalent for the PCR. In addition; no amplification was detected from DNAs of SGPV tested by the primer used for ORFV and veers versa in current applied assays. The results of the study suggest that; the presented RT-PCR provides a highly robust and sensitive method to detect SGPV and ORFV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where the two viruses co-circulate. To our knowledge, this is the first study to used the developed indoor new SYBR green IRT-PCR assay to identify of SGPV and ORFV and strong association between the PCR and RT-PCR using the same explicit primer set for each virus