The virus belongs to the genus of capripox of the family Poxviridae and has a double stranded genome of approximately 150 kbp. Lumpy skin disease virus (LSDV) is the causative agent of lumpy skin disease (LSD), a WOAH notifiable disease with significant socio-economic impacts on the local and international level. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains.
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