鼠轮状病毒荧光定量PCR检测方法的建立及初步应用

DEVELOPMENT OF REAL-TIME RT-PCR FOR DETECTION OF MURINE ROTAVIRUS

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。

To develop real-time PCR for detection of Murine rotavirus(MRV),one pair of primers and TaqMan probe were designed according to the genomic sequence of VP1gene of EB-C8/G16P strain(GenBank access No.KJ477127.1).The linearity,specificity and sensitivity of the method were optimized.The results showed an excellent linear relationship of standard curve,in which R2value reached up to0.99.Variation coefficient between batches was less than2%.The sensitivity of the TaqMan-probe real-time PCR was10copies/^L,i.e.100times higher than that of ordinary PCR method.The PCR method did not react with other mouse strains tested,indicating good specificity.Total246mouse samples were tested and all were negative.