Real time PCR for the detection of field isolates of lumpy skin disease virus in clinical samples from cattle

Lumpy skin disease caused by lumpy skin disease virus (LSDV, Capripoxvirus, Poxviridae ) is a capripoxviral disease with significant morbidity in cattle, which necessitates the development of reliable diagnostic tools in the context of live vaccine administration. OIE-recommended PCR assays target not only LSDV but also sheep pox virus and goat pox virus. Conventional PCR is prone to carry-over contamination, whereas real-time PCR offers more advantages, including prevention of amplicon carryover contamination post amplification. In this paper we report the development of a PCR real time assay for the detection of field isolates of lumpy skin disease virus in clinical samples from cattle. The specificity was validated against a panel of homologous and heterologous viruses retrieved from the strain depository of FGBI ARRIAH. The PCR assay was shown to be highly specific toward field LSDV. When tested in the presence of vaccine strain DNA and related capripoxviruses, no false-positive results were obtained. Using a series of 10-fold dilutions the assay proved to be highly sensitive with a detection limit of 0.21 lg TCD50/ml. The calculated efficiency of amplification was 98.6 %, with SD ranging from 0.11 to 0.33 over five orders of magnitude. The PCR assay was also validated on samples from experimentally inoculated bulls. The animals received a subcutaneous injection of a field LSDV and were tested for the presence of LSDV DNA in blood and nasal swab in comparison to PCR by D.C. Ireland и Y.S. Binepal (1998) (data not shown). Overall, the presented assay demonstrated high specificity and sensitivity and can be recommended as a diagnostic tool for the detection of field isolated of LSDV.