TaqMan-based real-time quantitative fluorescence PCR for detection of Orf virus

Contagious ecthyma, also known as scabby mouth or Orf, is a zoonosis, which is caused by the Orf virus (ORFV). Human contact with infected animals can cause cutaneous lesions. To prevent and control ORFV effectively, rapid detection method is very important and highly needed. Real-time quantitative fluorescence PCR (qPCR) assay is considered as a rapid techonology to detect ORFV, and has been used for clinical diagnosis and epidemiological investigation. In present study, we developed a TaqMan-based qPCR assay for detection of ORFV. Beacon Designer 7.9 was used to design specific primers and probes were based on the ORFV020 gene sequence of the virus (GenBank Accession No. KF666563.1). The method had no cross-reactions with other common bacteria and viruses, was highly specific; the sensitivity test result showed that it could detect 10 copies of ORFV genomic DNA, and was more sensitive than conventional PCR. Both intra-and inter-variabilities were less than 2%, indicating the high stability and repeatability of the method. Additionally, 99 clinical samples from sheep and goats with suspected contagious ecthyma were tested using the developed assay and conventional PCR. The results showed that the developed assay was more sensitive and faster than conventional PCR. It can be concluded that the assay was suitable for routine detection of the ORFV and the epidemiological investigation.