Development of real time PCR for diagnosis of capripoxviruses

Capripoxviruses cause economically important poxviral diseases in cattle, sheep and goats which are reportable to the World Organization for Animal Health. A sensitive, quick and specific confirmatory diagnosis is the foremost requirement for control and eradication of these diseases. In this study, TaqMan and SYBR green based quantitative polymerase chain reaction (qPCR) and conventional PCR assays targeting the P32 gene were developed for the detection of Capripoxvirus DNA in clinical specimens. Both conventional and real time assays used different primer sets. The conventional PCR assay used primer pairs which yielded amplicon of expected size (581 bp). The specificity of amplified P32 gene product was confirmed by its correct size and further by sequence analysis. Real time assay for Capripoxvirus has been developed and validated using SYBR green and TaqMan formats, which yielded a 72 bp product. Although both the conventional and real time assays were highly sensitive and specific for capripoxviruses however, the qPCR in both formats was 1000 times more sensitive than conventional PCR and was able to detect as low as 70 fg of viral DNA. This assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.