水体中诺如病毒富集技术的研究

Study on the technology of concentrating norovirus from water

目的评估不同实验条件下NanoCeram-PEG富集法对水中诺如病毒(NV)的富集效果,以获得较优的富集方法。方法构建诺如病毒阳性标准质粒,以此为模板,应用荧光定量RT-PCR方法绘制病毒定量标准曲线;将含诺如病毒粪便悬液加入纯净水中,在不同实验条件下经富集操作,富集液经荧光定量RT-PCR方法进行绝对定量并计算最终回收率,从而确定最优富集方法。结果通过病毒定量标准曲线确定荧光定量RT-PCR方法对水中诺如病毒的检测灵敏度为100 copy/μl。本实验通过正交设计方法考察了洗脱液p H值、洗脱速度和PEG浓度3个因素对最终病毒回收率的影响,最终确定在洗脱液流速为300 ml/min,洗脱液p H值为9.5,PEG6000浓度为13%的条件下病毒回收率最高,达到26.55%。结论证明NanoCeram-PEG富集法具有较好的病毒回收率,加以发展将为水源性诺如病毒疫情的溯源提供可靠的实验室依据。

Objective Evaluate the enrich efficiency of the Nano Ceram-PEG concentration method for norovirus from water under different experimental conditions,so as to obtain the optimum concentration conditions. Methods The constructed positive norovirus positive plasmid was used for generating standard curve by fluorescent quantitation RT-PCR method for quantitative analysis of norovirus. Fecal suspension which contains norovirus was seeded to purified water. Virus in the water then underwent concentration procedure under different experimental conditions,and after detecting by fluorescent quantitation RT-PCR,the viral copies of the final elute and accordingly the recovery rate of virus were determined based on the plotted standard curve. Finally,the optimum condition should be concluded from the recovery rate results. Results According to plotted standard curve,the sensitivity of fluorescent quantitation RT-PCR for norovirus in water was 100 copy / μl. This study investigated the effect of three factors（ eluent p H,elution rate and PEG concentration） on final recovery rate of virus by orthogonal test and among the conditions examined,the condition of eluent flow rate of 300 ml / min,the eluent of p H 9. 5 and PEG6000 concentration of 13% showed the best virus recovery efficiency（ 26. 55%）. Conclu sion Nano Ceram-PEG concentration method manifests a sound virus recovery efficiency. If developed,it will be a reliable laboratory basis for tracing source of outbreak of waterborne norovirus.