• 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

牛病毒性腹泻/黏膜病病毒荧光定量PCR检测方法的建立

A RT-PCR Assay for Quantitative Detection of Bovine Viral Diarrhea Virus

  • 摘要:
      目的  设计一种能够快速检测出牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)的方法。
      方法  依据GenBank公布的BVDV 5′UTR基因属于特异性保守区域的前提,构建具有特异性的探针和引物,以体外转录病毒RNA作为绝对定量标准品。对荧光定量RT-PCR方法的各反应条件进行优化,创建BVDV荧光定量PCR检测方法。
      结果  5.026 7 copies·μL−1是此检测方法的最低下限值,该方法拥有良好的重复性,变异系数在组内、组间均<1%。特异性较高,对其他病毒核酸的扩增均为阴性,如猪瘟病毒、口蹄疫病毒等。
      结论  建立的BVDV荧光定量PCR方法敏感性高、特异性强、重复性好,为牛病毒性腹泻的早期诊断提供了重要的技术支撑。

     

    Abstract:
      Objective   A rapid detection and quantification method for bovine viral diarrhea virus (BVDV) was established.
      Method   For the methodology development, specific primers and probes were designed based on the target regions of the BVDV 5′UTR gene published by GenBank. The RNA of in vitro transcription viruses was used as the absolute quantitative standard. Reaction conditions of the fluorescent quantitative RT-PCR were optimized.
      Result  The newly developed assay had a minimum detection limit of 5.0267copies/μL and an intragroup variation coefficient of less than 1% with high repeatability and specificity. Other than BVDV, it amplified no viral nucleic acids of viruses such as swine fever and foot-and-mouth diseases.
      Conclusion  The established fluorescence quantitative RT-PCR method was highly sensitive, specific, and repeatable in detecting and quantifying BVDV. It appeared appropriate for early diagnosis of bovine viral diarrhea.

     

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