Development and validation of a real-time RT-PCR assay for detection of Bluetongue virus
- 1Research Institute for Biological Safety Problems, National Holding QazBioPharm, Gvardeyskiy, Kazakhstan
- 2National Reference Center for Veterinary, Almaty, Kazakhstan
- 3Kazakhstan Limited Liability Partnership Scientific and Production Center DiaVak-ABN, Almaty, Kazakhstan
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.
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BibTeX
@article{koshemetov2026development,
title = {{Development and validation of a real-time RT-PCR assay for detection of Bluetongue virus}},
author = {Zh. K. Koshemetov and A. A. Issakhan and K. A. Shorayeva and Ye. A. Bulatov and N. K. Orazymbetova and A. K. Abubakirova and K. T. Sultankulova and M. Zh. Kaukarbayeva and Zh. B. Kondibayeva and B. K. Umuraliyev and O. N. Serikbayov and M. S. Seisenbayeva and A. K. Adalbekova and K. T. Maikhin and A. A. Abishov and K. D. Zhugunissov},
journal = {Frontiers in Veterinary Science},
year = {2026},
month = oct,
doi = {10.3389/fvets.2026.1859742},
url = {https://doi.org/10.3389/fvets.2026.1859742},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development and validation of a real-time RT-PCR assay for detection of Bluetongue virus
AU - Zh. K. Koshemetov
AU - A. A. Issakhan
AU - K. A. Shorayeva
AU - Ye. A. Bulatov
AU - N. K. Orazymbetova
AU - A. K. Abubakirova
AU - K. T. Sultankulova
AU - M. Zh. Kaukarbayeva
AU - Zh. B. Kondibayeva
AU - B. K. Umuraliyev
AU - O. N. Serikbayov
AU - M. S. Seisenbayeva
AU - A. K. Adalbekova
AU - K. T. Maikhin
AU - A. A. Abishov
AU - K. D. Zhugunissov
PY - 2026
DA - 2026/10/21
JO - Frontiers in Veterinary Science
DO - 10.3389/fvets.2026.1859742
UR - https://doi.org/10.3389/fvets.2026.1859742
AB - 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.
SN - 2297-1769
ER -