Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses

Area of Science:

• Veterinary Virology
• Molecular Diagnostics
• Epidemiology

Background:

• Capripoxvirus (CaPV) includes sheep poxvirus (SPPV), goatpoxvirus (GTPV), and lumpy skin disease virus (LSDV), causing significant livestock diseases.
• Current diagnostic methods struggle to differentiate CaPV strains, particularly SPPV and GTPV, hindering accurate disease surveillance and management.
• Conflicting host specificity data for SPPV and GTPV necessitates advanced genotyping tools for precise identification.

Purpose of the Study:

• To develop a rapid, species-specific real-time polymerase chain reaction (PCR) assay for Capripoxvirus (CaPV) detection and genotyping.
• To utilize unique molecular markers within the G-protein-coupled chemokine receptor (GPCR) gene for CaPV strain differentiation.
• To facilitate simultaneous detection, quantitation, and genotyping of CaPV isolates.

Main Methods:

• Development of a real-time PCR assay targeting unique molecular markers in the CaPV GPCR gene.
• Utilized dual hybridization probes for simultaneous detection and quantitation.
• Employed fluorescence melting curve analysis (FMCA) to differentiate CaPV strains based on melting point temperature (Tm) variations.

Main Results:

• The developed real-time PCR assay is species-specific and capable of differentiating CaPV strains.
• The assay demonstrated high sensitivity with low intra- and inter-run variation.
• Strain differentiation was achieved by analyzing melting point temperatures (Tm) from FMCA.

Conclusions:

• This real-time PCR assay provides a significant advancement for Capripoxvirus (CaPV) diagnosis.
• The assay enables rapid genotyping and gene-based classification of viral strains, improving epidemiological understanding.
• Unequivocal identification of CaPV isolates is facilitated, aiding in better management of capripox outbreaks.