Bluetongue is a vector-borne disease caused by Bluetongue virus, an orbivirus transmitted by biting midges of the genus Culicoides. The virus has a geographically broad distribution and has affected livestock regions in Europe, Asia, North and South America, Australia and New Zealand. Kazakhstan’s substantial population of susceptible cattle, sheep and goats could facilitate transmission if the virus were introduced, while outbreaks can impose economic costs, restrict livestock exports and affect a country’s epizootic status. The study was designed as a laboratory assay-development and validation study, rather than an intervention trial or an assessment of whether use of the test reduced disease transmission.

Timely molecular diagnosis is particularly important where bluetongue circulates or where infection may be introduced. Serological assays such as enzyme-linked immunosorbent assays can be difficult to interpret in endemic settings because antibodies may reflect prior infection or vaccination rather than current viral activity. Real-time RT-PCR instead detects viral RNA and can identify low concentrations of the pathogen, including in animals without clear clinical signs. The investigators sought a locally developed test that combined sensitivity, specificity and reproducibility with the practical requirements of veterinary laboratories and surveillance programs. Their target was the VP7 gene in genome segment 10, selected after sequence comparisons identified conserved regions potentially detectable across BTV serotypes.

Four primer–probe sets were designed with Primer Express 3.0.1, Oligo Analyzer and Primer-BLAST, with sequence alignment performed in ClustalW and MEGA 10. The oligonucleotides contained degeneracy codes to accommodate variation among BTV sequences, and specificity was checked against GenBank through BLAST. Each set was evaluated in conventional and real-time RT-PCR. Conventional amplification with BTV10/1, BTV-VP7 and BTV-VP7/1 produced products of the expected size, while BTV10S/2 showed weak amplification. Quantification did not support using every nominally functional set: BTV10S/2 yielded cycle-threshold values near 40, and BTV-VP7/1 failed to produce detectable amplification in the reported control comparisons.

The BTV10/1 set was selected for further development because it generated stable signals with low Ct values in both plasmid-DNA and BTV RNA controls, reported as 17.44 ± 0.32 and 19.70 ± 0.28, respectively. Its expected amplicon was 112 base pairs. The investigators optimized probe concentration over a range of 50 to 250 nanomolar and found that 150–250 nanomolar produced the most stable amplification. The final mixture was reported to contain 1 unit of Taq polymerase, 0.2 millimolar deoxynucleoside triphosphates, 2.5 millimolar magnesium ions, 400–600 nanomolar primers and a 150–200 nanomolar probe. Reactions ran on a Rotor-Gene Q system, with reverse transcription at 50 °C for 30 minutes, initial denaturation at 95 °C for 15 minutes and 45 cycles of 95 °C for 15 seconds and 55 °C for 30 seconds.

Fluorescence was measured in the FAM channel during annealing and extension, and a sample was classified as positive when its Ct value was below 38. Samples with Ct values of 38 or above, or without amplification, were treated as negative. Analytical sensitivity was assessed with tenfold dilutions of a plasmid containing a 121-base-pair fragment of the BTV genome. The assay consistently detected the 3-picogram dilution, whereas the 0.3-picogram dilution was not reliably detected: replicate measurements at that level gave Ct values of 40.2 and two non-detections. The standard curve showed a strong linear relationship between Ct and the serial dilutions, with a coefficient of determination of 0.988 and an estimated amplification efficiency of 91.2%. These data define the reported analytical limit in mass units of the plasmid target rather than as a directly measured number of RNA copies.

Analytical specificity was evaluated against BTV and six heterologous animal viruses representing the Rhabdoviridae, Paramyxoviridae, Herpesviridae, Retroviridae and Coronaviridae families. The panel comprised rabies virus, peste des petits ruminants virus, canine distemper virus, Aujeszky’s disease virus, bovine leukemia virus and infectious bronchitis virus. Only BTV produced an exponential fluorescence signal; none of the heterologous viruses amplified, and the nuclease-free-water negative control also remained negative. This panel provides evidence against cross-reactivity with the viruses tested, although it does not establish exclusion of every possible non-target agent or every BTV serotype present in nature.

For clinical validation, the researchers analyzed 61 blood samples from cattle and small ruminants collected from Zhetysu, Almaty, Turkestan, Zhambyl, Pavlodar and North Kazakhstan regions. Most samples were negative by both the new assay and the commercial PCR-Bluetongue-Factor reference test. Positives were found mainly in small ruminants in Zhetysu, Turkestan and Zhambyl, whereas positive results in cattle were sporadic; all samples from Pavlodar and North Kazakhstan were negative under both methods. The new assay and reference test each classified eight samples as positive and 53 as negative, but each also produced one apparently discordant positive result. In the 2 × 2 agreement analysis, the new assay had seven true positives, one false positive, one false negative and 52 true negatives relative to the commercial assay.

Those concordance data yielded a relative sensitivity of 87.5%, calculated from seven of eight reference-positive samples, and relative specificity of 98.1%, calculated from 52 of 53 reference-negative samples. Overall agreement in that analysis was 96.7%, and Cohen’s kappa was 0.86, described by the researchers as almost perfect agreement. In a separate presentation of diagnostic performance, the authors reported sensitivity of 80%, specificity of 98.14%, positive and negative predictive values of 88.89% and 96.36%, and overall accuracy of 95.31% for the developed assay. The commercial test was reported to have sensitivity of 72.7% and specificity of 98.14%; both assays had a positive predictive value of 88.89%, while the commercial test’s negative predictive value was 94.64%, compared with 96.36% for the developed assay. The two discrepant samples were one cattle specimen from Almaty’s Uygur district and one small-ruminant specimen from Zhambyl’s Sarysu district. The investigators attributed the differences tentatively to low viral loads near the detection limit, where stochastic distribution of RNA molecules can make amplification outcomes variable.

The study concludes that the optimized assay offers a sensitive, specific and reproducible means of detecting BTV RNA for veterinary diagnosis and epidemiological surveillance. Its 3-picogram analytical detection limit, negative results for all six heterologous viruses tested and close agreement with a commercial assay support further operational evaluation. Important limitations remain: the clinical sample set was small, included only 61 specimens, and was not interlaboratory validated. The samples also represented a limited set of regions, animal populations and monitoring collections, while the specificity panel did not cover the full range of pathogens that might occur in livestock. The authors therefore called for larger studies across more diverse regions and additional validation before broader implementation, while noting that continued primer-sequence review may be needed as BTV genetic variation and potentially new serotypes emerge.

Subject of Research: Laboratory development and clinical validation of a real-time RT-PCR assay for detecting Bluetongue virus RNA in cattle and small-ruminant blood samples from Kazakhstan

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

Article References: Koshemetov, Z. K., Issakhan, A. A., Shorayeva, K. A., Bulatov, Y. A., Orazymbetova, N. K., Abubakirova, A. K., Sultankulova, K. T., Kaukarbayeva, M. Z., Kondibayeva, Z. B., Umuraliyev, B. K., Serikbayov, O. N., Seisenbayeva, M. S., Adalbekova, A. K., Maikhin, K. T., Abishov, A. A., & Zhugunissov, K. D. (2026). Development and validation of a real-time RT-PCR assay for detection of Bluetongue virus. Frontiers in Veterinary Science, 13, Article 1859742. https://doi.org/10.3389/fvets.2026.1859742

Image Credits: AI Generated

DOI: 10.3389/fvets.2026.1859742

Keywords: Bluetongue virus, real-time RT-PCR, veterinary diagnostics, livestock surveillance, molecular assay validation, VP7 gene, diagnostic sensitivity, diagnostic specificity, cattle, small ruminants, Kazakhstan, Development