Development of a Cost-Effective Method for Capripoxvirus Genotyping Using Snapback Primer and dsDNA Intercalating Dye

Abstract

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies.

Epidemiological and Molecular Studies on Lumpy Skin Disease Outbreaks in Turkey during 2014-2015

“…The real‐time PCR high‐resolution melt (HRM) assay that can distinguish each type of CaPV was used for genotyping. Primers designed to target the 30 kDa RNA polymerase subunit (RPO30) gene of CaPVs were used for genotyping field viruses (Gelaye et al., ). GTPV Turkey/98 Denizli, SPPV Turkey/2015‐Akşehir and LSDV Turkey/2014‐01 were used as positive controls.…”

Lumpy Skin Disease: Global and Turkish Perspectives

“…PCR and real time PCR have been used for molecular detection of LSDV in samples taken from skin lesions, blood, saliva, milk and semen [10,11,[14][15][16][17][18]. Viral DNA can be detected in the skin lesions by PCR for 42 days [9] and 92 days [14] after experimental infection.…”

Scientific Opinion on sheep and goat pox

“…A comparative analysis of the complete genome of five SPPV and GTPV isolates, including three pathogenic field isolates and two attenuated vaccine viruses, has highlighted differences between SPPV and GTPV (and LSDV) at the genome level (Tulman et al, 2002). These differences in genome sequence have been used to develop real-time PCR-based diagnostic techniques, enabling classification of the strains as SPPV, GTPV or LSDV (Gelaye et al, 2013;Lamien et al, 2011b;Murray et al, 2013). This initial evidence, based on seven whole-genome sequences, indicates that SPPV and GTPV, although highly similar, are phylogenetically distinct viral species (Tulman et al, 2002).…”

“…A gel-based PCR for the differentiation of SPPV and GTPV has been described (Lamien et al, 2011a), as has a novel method based on snapback primers (Gelaye et al, 2013). As mentioned above, these methods are not as fast and sensitive as real-time PCR.…”