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Background <p>Bovine ephemeral fever (BEF) is a significant disease in nations such as India, where there is a large bovine population and environmental conditions which foster the proliferation of blood-feeding insects. Effective control and prevention of the disease necessitate early and precise detection of bovine ephemeral fever virus (BEFV), the causative agent of BEF. In the present study, a TaqMan real time PCR assay with high sensitivity and specificity was developed for accurate detection of BEFV.</p> Methods and results <p>The glycoprotein (G) gene of BEFV was targeted for the assay development. A positive construct of the G gene was developed by identifying the conserved regions through the alignment of the available G gene sequences from GenBank. The primers and probes had been designed in-house and synthesised commercially. The TaqMan probe-based assay was standardized using the gene construct as a positive control. The developed assay demonstrated an exceptional sensitivity for the detection and quantification of viral genomes, capable of identifying as few as 24 copies of the genome with a R² value of 0.9998 by standard curve analysis. The developed assay demonstrated a sensitivity that was 100 times greater than the conventional PCR method and showed remarkable efficiency for the BEFV detection using field blood samples from both suspected and healthy animals.</p> Conclusion <p>The developed assay showed high sensitivity and specificity in clinical applicability analysis. This real-time PCR-based assay would offer a reliable and adaptable diagnostic tool for the rapid and precise identification of BEFV in disease diagnosis, research, and field surveillance applications.</p>

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Development of a TaqMan based real-time PCR assay for the detection of bovine ephemeral fever virus

  • 期刊论文

  • 发表日期：

    2025年10月17日

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  • Shivam Solanki,
  • M. Manu,
  • Adarsh Mishra,
  • Mousumi Bora,
  • Vishal Mahajan,
  • Kuldip Gupta

摘要

Background

Bovine ephemeral fever (BEF) is a significant disease in nations such as India, where there is a large bovine population and environmental conditions which foster the proliferation of blood-feeding insects. Effective control and prevention of the disease necessitate early and precise detection of bovine ephemeral fever virus (BEFV), the causative agent of BEF. In the present study, a TaqMan real time PCR assay with high sensitivity and specificity was developed for accurate detection of BEFV.

Methods and results

The glycoprotein (G) gene of BEFV was targeted for the assay development. A positive construct of the G gene was developed by identifying the conserved regions through the alignment of the available G gene sequences from GenBank. The primers and probes had been designed in-house and synthesised commercially. The TaqMan probe-based assay was standardized using the gene construct as a positive control. The developed assay demonstrated an exceptional sensitivity for the detection and quantification of viral genomes, capable of identifying as few as 24 copies of the genome with a R² value of 0.9998 by standard curve analysis. The developed assay demonstrated a sensitivity that was 100 times greater than the conventional PCR method and showed remarkable efficiency for the BEFV detection using field blood samples from both suspected and healthy animals.

Conclusion

The developed assay showed high sensitivity and specificity in clinical applicability analysis. This real-time PCR-based assay would offer a reliable and adaptable diagnostic tool for the rapid and precise identification of BEFV in disease diagnosis, research, and field surveillance applications.