贝类中诺如病毒SYBR Green I实时定量RT-PCR检测方法研究

Studies on SYBR Green I Real-time Fluorescence Quantitative RT-PCR for Detecting Norovirus in Shellfish

将诺如病毒(Noroviruses,NVs)RT-PCR特异产物纯化后与pGEM-T载体连接并转化至感受态细胞DH5α,经测序鉴定,提取阳性克隆质粒DNA,梯度稀释后作为实时荧光定量PCR的标准质粒。使用SYBR Green I染料建立检测贝类中NVs的实时荧光定量RT-PCR方法。结果表明,本方法标准曲线线性范围可达6个数量级(108～103拷贝),R2为0.9983,比常规RT-PCR方法敏感100倍,且特异性良好,与贝类中的轮状病毒、甲肝病毒均不反应;批内和批间重复性良好,变异系数(CV)分别在0.28%～4.03%和1.47%～5.53%范围。该方法具有简便、敏感、高效、稳定等优点,可用于水产品中NVs的定量检测。

The target gene of NVs was amplified by RT-PCR,after purified,the PCR products were cloned into pGEM-T vector,and transformed into competent E.Coli.DH5α.The plasmid identified by sequencing and the DNA of recombinant vector was extracted,diluted and used as standards.A Real-time RT-PCR method was developed for detection of NVs in shellfish using the SYBR Green I.The result shows that constructed curve had wide linear range of six orders at least and correlation coefficient was 0.9983;the developed real-time FQ RT-PCR was 100 times more sensitive than the conventional RT-PCR for detection of NVs.Other virus,such as Rotavirus（RV） and Hepatitis A virus（HAV） were not reacted.The coefficient of variation for both intra-experimental reproducibility and inter-experimental reproducibility ranged from 0.28% to 4.03% and 1.47% to 5.53% respectively.The result showed that the Real-time FQ RTPCR was simple,sensitive,efficient,stable,and could be used as quantitative detection of NVs in aquatic product.