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根据鼠轮状病毒(Murine rotavirus,MRV)EB-C8/G16P毒株VP1基因(GenBank登录号:KJ477127.1),设计引物和Taq Man探针,确定标准曲线,建立MRV荧光定量PCR方法,检测其特异性、敏感性和稳定性。结果显示,建立的MRV荧光定量PCR方法标准曲线的线性关系良好,R2值可达0.99;最低可检测到10 copies/μL,是常规PCR的100倍;与常见的小鼠病毒株均无特异性扩增;批内和批间变异系数均小于2%,表明该方法重复性好。将建立的荧光定量PCR初步对246份小鼠肠道样品病料进行检测,结果均为阴性。本研究建立的MRV荧光定量PCR方法特异性、灵敏性良好,可为鼠轮状病毒的流行病学调查、检测以及制定国家标准和地方标准提供可靠的数据和适用的监测方法。
According to the VP1 gene (GenBank accession number: KJ477127.1) of Murine rotavirus (MRV) EB-C8 / G16P strain, primers and TaqMan probe were designed to determine the standard curve, and MRV fluorescence quantitative PCR method was established to detect Its specificity, sensitivity and stability. The results showed that the established standard curve of MRV fluorescence quantitative PCR had a good linear relationship with a R2 value of 0.99 and a minimum detectable concentration of 10 copies / μL, which was 100 times higher than that of the conventional PCR. The coefficients of variation within and between batches were all less than 2%, indicating that the method was reproducible. The established fluorescence quantitative PCR preliminary detection of 246 samples of intestinal tract disease in mice, the results were negative. The MRV fluorescence quantitative PCR method established in this study is specific and sensitive and can provide reliable data and applicable monitoring methods for the epidemiological investigation and detection of murine rotavirus as well as the development of national and local standards.