纯净水中病毒三种富集方法的比较

Comparison of three methods for virus enrichment from purified water

目的比较膜吸附法、膜吸附-PEG法和膜吸附-超滤法对纯净水中病毒的富集效果,并对优选的富集方法的实验条件进行比对,旨在寻找高效的方法,为其他水体中病毒富集检测提供技术借鉴。方法本文以MS2噬菌体为目标病毒,制作高、低浓度水样,建立逆转录-荧光定量聚合酶链式反应(RT-qPCR)方法的标准曲线,进行定量检测。使用膜吸附法、膜吸附-PEG法和膜吸附-超滤法富集水样,并比较不同过滤膜、不同超滤管及两种核酸提取方法的回收率,选择最适方法。结果膜吸附-超滤法、膜吸附-PEG法和膜吸附法回收率均值分别为19.80%±12.19%、12.71%±9.09%和9.05%±4.89%,各因素对回收率的影响顺序为:膜吸附-超滤法>膜吸附-PEG法>膜吸附法;高浓度>低浓度。混合纤维素酯膜的富集效果优于尼龙膜。100 K和50 K的超滤管富集效果无差异。硅胶离心柱法和磁珠法核酸提取效果无差异。结论通过二次富集可提高富集效率,膜吸附-超滤法优于另两种方法,且其操作简单、耗时短、对设备依赖性低,易于推广。膜吸附-超滤法的实验条件仍可优化、完善,拓展其在不同水体和不同病毒富集中的应用。

Objective To compare the effects and experimental conditions of membrane adsorption method,membrane adsorption-PEG method and membrane adsorption-ultrafiltration method on virus enrichment in purified water.Methods MS2 bacteriophage was used as target virus to prepare water samples with high and low concentrations,and the standard curve of reverse transcription-fluorescence quantitative polymerase chain reaction(RT-qPCR)was established for quantitative detection.Water samples were enriched by membrane adsorption method,membrane adsorption-PEG method and membrane adsorption-ultrafiltration method.The recoveries of different filtration membranes,different ultrafiltration tubes and two nucleic acid extraction methods were compared.Results The average recoveries of membrane adsorptionultrafiltration method,membrane adsorption-PEG method and membrane adsorption method were 19.80%±12.19%,12.71%±9.09%and 9.05%±4.89%.The order of influencing factors on the recoveries was:membrane adsorptionultrafiltration method>membrane adsorption-PEG method>membrane adsorption method.High concentration>low concentration.The effect of virus enrichment of mixed cellulose ester membrane was better than nylon membrane.There was no difference in the enrichment effect of ultrafiltration tube at 100 and 50 K,and the nucleic acid extraction effect of silica gel centrifugation column method and magnetic bead method.Conclusion The enrichment efficiency can be improved by secondary enrichment.Membrane adsorption-ultrafiltration method is simple to operate,short time consuming,low dependence on equipment,and easy to be popularized.The experimental conditions of membrane adsorption-ultrafiltration can be optimized and improved to expand the application of different water bodies and different virus enrichment.