• 中文核心期刊
  • CSCD来源期刊
  • 中国科技核心期刊
  • CA、CABI、ZR收录期刊
ZHAO R T, Temuerbagen, GUO Y, et al. TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus [J]. Fujian Journal of Agricultural Sciences,2023,38(7):851−856. DOI: 10.19303/j.issn.1008-0384.2023.07.011
Citation: ZHAO R T, Temuerbagen, GUO Y, et al. TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus [J]. Fujian Journal of Agricultural Sciences,2023,38(7):851−856. DOI: 10.19303/j.issn.1008-0384.2023.07.011

TaqMan qRT-PCR Assay for Detecting Bovine Kobuvirus

  •   Objective  A rapid, accurate qRT-PCR method for detecting bovine kobuvirus (BKoV) was established and clinically tested for application.
      Method  A pair of specific primers and a probe were designed and synthesized according to the 3D gene sequence of BKoV published on GenBank for the establishment of a TaqMan fluorescence qRT-PCR assay. The reaction system of the analytic was optimized prior to verification on clinical specimens.
      Result  The optimal upstream and downstream primers were 300 nmol·L−1 in concentration, and the applied probe 400 nmol·L−1. A linearity with a correlation coefficient of R2= 0.999 was achieved in the range of 1×101−1×108 copies·μL−1. The amplification efficiency reached 103%. The assay demonstrated a high specificity among the pathogens related to diarrhea in calves, a high sensitivity with the minimum detection limit of 1×101 copies·μL−1 on BKoV plasmid standard, in comparison to that of 1×102 copies·μL−1 by conventional RT-PCR, and a high repeatability with a coefficient of variation of less than 3% within and between groups. In 37 bovine fecal samples collected from different pastures in Inner Mongolia from March to May 2021, the assay showed a positive detection rate of 24.3%. The calculated viral loads using the standard curve had an average on the feces from diarrheal animals at 3.7×105 copies·μL−1 and at 8.65×103 copies·μL−1 on the specimens from healthy calves.
      Conclusion  The newly developed TaqMan fluorescent qRT-PCR assay was specific, repeatable, and sensitive in detecting BKoV. It could be applied for clinic diagnosis and epidemiological investigation on the disease.
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