Development and validation of a real-time RT-PCR assay for detection of Bluetongue virus

Abstract

Bluetongue virus (BTV) poses a significant threat to livestock production, highlighting the need for highly sensitive and specific diagnostic tools for its detection. The aim of this study was to develop and validate a real-time reverse transcription polymerase chain reaction (RT-PCR) assay for the sensitive detection of BTV RNA in field samples. Four primer–probe sets targeting conserved regions of the viral genome were designed and evaluated. Among them, the BTV10S/1 primer–probe set demonstrated the highest amplification efficiency, providing stable and reproducible detection results. Reaction conditions and the composition of the PCR mixture were optimized. The analytical sensitivity of the developed assay reached 3 pg. of plasmid DNA. High specificity was confirmed by the absence of cross-reactivity with RNA from heterologous animal viruses. Clinical validation using 61 field samples collected from cattle and small ruminants showed a diagnostic sensitivity of 80%, specificity of 98.14%, and an overall diagnostic accuracy of 95.31%. The developed assay enables reliable and early detection of BTV in field conditions and can be applied in veterinary practice for epidemiological surveillance and disease control. Implementation of this method contributes to improved biosafety by facilitating timely detection and limiting the spread of infection.