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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 To evaluate the enrichment effect of NanoCeram-PEG enrichment method on Norovirus (NV) in water under different experimental conditions in order to obtain a better enrichment method. Methods Norovirus positive standard plasmids were constructed and used as a template to quantify the quantitative standard curve of virus by fluorescence quantitative RT-PCR method. The norovirus suspension was added into purified water and enriched by different experimental conditions. The collection volume was quantified by fluorescence quantitative RT-PCR and the final recovery was calculated to determine the optimal enrichment method. Results The detection limit of norovirus in water was 100 copy / μl by fluorescence quantification RT-PCR method. In this experiment, we investigated the influence of pH value, elution rate and PEG concentration on the final virus recovery rate by orthogonal design method. Finally, the eluent p Under the conditions of H value of 9.5 and PEG6000 concentration of 13%, the virus recovery was the highest, reaching 26.55%. The conclusion proves that NanoCeram-PEG enrichment method has a good virus recovery rate, and its development will provide a reliable laboratory basis for traceability of waterborne norovirus virus.