Abstract
Outbreaks of the three capripox virus species, namely lumpy skin disease virus, sheeppox virus, and goatpox virus, severely affect animal health and both national and international economies. Therefore, the World Organization for Animal Health (OIE) classified them as notifiable diseases. Until now, discrimination of capripox virus species was possible by using different conventional PCR protocols. However, more sophisticated probe-based real-time qPCR systems addressing this issue are, to our knowledge, still missing. In the present study, we developed several duplex qPCR assays consisting of different types of fluorescence-labelled probes that are highly sensitive and show a high analytical specificity. Finally, our assays were combined with already published diagnostic methods to a diagnostic workflow that enables time-saving, reliable, and robust detection, differentiation, and characterization of capripox virus isolates.
Highlights
Differentiation of Capripox VirusThe three species lumpy skin disease virus, goatpox virus, and sheeppox virus form the genus Capripoxvirus within the family Poxviridae [1]
Different PCR detection systems have been developed over the years, ranging from conventional gel-based assays combined with agarose gel electrophoresis [20] or with restriction fragment length polymorphism (RFLP) analyses [21] through loop-mediated isothermal amplification (LAMP) assays [22,23] and high-resolution melting curve analyses [24] to real-time quantitative PCR assays using intercalating dyes [20] or fluorescence-labelled probes [6,25,26]
The first focused on the enhanced green fluorescent protein (EGFP)—DNA as heterologous internal control added during the extraction process [32] and the second used the β-Actin housekeeping DNA [33]
Summary
The three species lumpy skin disease virus, goatpox virus, and sheeppox virus form the genus Capripoxvirus within the family Poxviridae [1]. Capripox viruses (CaPVs), mainly infecting cattle, goats, and sheep, respectively [2,3], are described as the most serious poxvirus diseases of domestic animals [2,4,5,6]. Due to severe production losses caused by. CaPV outbreaks (e.g., decreased growth rate and mass loss, decreased milk yield, damage to hide and skin as well as temporary or permanent infertility in bulls), outbreaks of these diseases have a massive impact on national as well as global economies [2,3,4,7,8,9,10]. Organization for Animal Health (OIE) [11]. Different PCR detection systems have been developed over the years, ranging from conventional gel-based assays combined with agarose gel electrophoresis [20] or with restriction fragment length polymorphism (RFLP) analyses [21] through loop-mediated isothermal amplification (LAMP) assays [22,23] and high-resolution melting curve analyses [24] to real-time quantitative PCR (qPCR) assays using intercalating dyes [20] or fluorescence-labelled probes [6,25,26]
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