• 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

猪A群轮状病毒TaqMan荧光定量RT-PCR检测方法的建立与运用

Establishment and Application of TaqMan Fluorescence Quantitative RT-PCR Detection Method for Porcine Rotavirus Group A

    摘要
  • 摘要:
    目的 建立一种猪A群轮状病毒（porcine rotavirus group A, PoRV A）快速检测方法，用于PoRV检测和流行病学调查。
    方法 参考GenBank中猪A群轮状病毒（PoRV A）VP6基因序列（登录号MT025937.1、OP978242.1、PP566178.1）设计特异性引物和探针，优化反应体系中引物和探针的浓度，建立TaqMan RT-qPCR检测方法，并通过特异性、灵敏性和重复性的结果以及临床应用对该方法进行评价。
    结果 该方法可特异性扩增PoRV核酸，最低检出限度为27.0 copies·μL−1，灵敏度高于普通RT-PCR 100倍；与猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）、猪德尔塔冠状病毒（porcine deltacorona virus, PDCoV）、猪传染性胃肠炎病毒（transmissible gastroenteritis of swine, TGEV）核酸均无交叉反应；组内和组间变异系数均小于1.10%，重复性好。151份疑似PoRV的临床样品使用RT-qPCR进行检测，结果显示检出率为42.38%（64/151），优于常规RT-PCR的检出率（33.11%，50/151）。
    结论 本研究基于猪A群轮状病毒VP6基因，建立了适用于PoRV A检测及其流行病学调查的TaqMan实时荧光定量PCR检测方法，具有灵敏度高、特异性强、重复性好等优势，为猪轮状病毒检测和流行病学调查提供了技术手段。

     

    Abstract:
    Objective  Establish a rapid detection method of porcine rotavirus group A(PoRV A)for pathogen monitoring and epidemiological investigation.MethodThis study designed specific primers and probes based on the VP6 gene sequences of PoRV A in GenBank (accession numbers MT025937.1,OP978242.1 and PP566178.1), optimized the reaction system, established a TaqMan RT-qPCR detection method, and evaluated the method through specificity, sensitivity, and repeatability results as well as its clinical application.
    Results This method can specifically amplify PoRV nucleic acid, with a minimum detection limit of 27.0 copies/μL and a sensitivity 100 times higher than RTPCR; There is no cross reactivity with the nucleic acid of PEDV, TGEV and PDCoV; This method has good repeatability, and the variation coefficients value of intra-group and inter-group are below 1.10%.; 151 clinical samples suspected of PoRV were detected using RT-qPCR, and the results showed a detection rate of 42.38%(64/151), which was better than the detection rate of conventional RT-PCR (33.11%, 50/151).
    Conclusion The TaqMan real-time fluorescent quantitative PCR detection method based on the VP6 gene of Group A porcine rotavirus in this study was established, which is suitable for PoRV detection and epidemiological investigations. The method offers high sensitivity, strong specificity, and good repeatability, providing a reliable technical means for the detection and epidemiological study of porcine rotavirus.

     

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