饮用水中病毒浓缩与检测方法的建立及应用

The establishment and application of the method with virus concentration and detection in drinking water

目的建立饮用水中病毒浓缩与检测方法并对实际水样进行人腺病毒污染状况监测。方法以f2噬菌体为水中肠道病毒代表，投加于受试水样中，评价NanoCeram滤芯对原水和饮用水初次浓缩及不同浓度PEG8000对初次浓缩液进行二次浓缩的效果。采用T以克隆制备人腺病毒荧光定量PCR标准品，对所得质粒测序，应用Blast序列类似性检索工具将其与目的基因片段进行一致性检测，建立荧光定量PCR检测人腺病毒的方法。应用上述方法，于2011年对武汉两家自来水厂原水和饮用水采用NanoCeram滤芯进行现场初次浓缩，聚乙二醇（PEG）／NaCl法进行二次浓缩，提取浓缩液中病毒DNA后进行荧光定量PCR检测，监测原水和饮水中人腺病毒污染状况。结果所建立的NanoCeram滤芯初次浓缩法对原水中的f2噬菌体回收率为（51．63±26．60）％，对饮用水中的￡噬菌体回收率为（50．27±14．35）％。当PEG8000浓度达到0．13kg／L时，二次浓缩回收率可达（90．09±10．50）％。所构建的人腺病毒荧光定量PCR标准品与目的基因片段序列一致度为99％，可用于人腺病毒的绝对定量。2011年，武汉市两家自来水厂原水中的人腺病毒浓度范围为（4．13×103～2．20×103）拷贝／L，出厂水中的人腺病毒浓度范围为（5．57×10。～7．52×105）拷贝／L，饮用水处理过程对人腺病毒的清除率为（75．49±11．71）％。结论NanoCeram滤芯联合PEG／NaCl法可对水环境中病毒进行有效浓缩，应用所建立的荧光定量PCR法检测发现自来水厂原水中存在人腺病毒，目前的水处理措施不能将其完全去除。

Objective This study aimed to construct an effective method to concentrate and detect virus in drinking water, and human adenovirus pollution status in actual water samples was monitored by constructed method. Methods The concentration efficient of NanoCeram filter for the first concentration with source water and drinking water and the coneentration efficient of the different concentrations of PEG 8000 for the second concentration were assessed by spiking f2 bacteriophage into water samples. The standard of human adenovirus for real-time PCR was constructed by T-A clone. The plasmid obtained was identified through sequence analyzing and consistency check comparing to target gene fragment was conducted by using blast algorithm, Then,real-time PCR was constructed to quantify the concentration of human adenovirus using the plasmid as standard. Water samples were concentrated by using NanoCeram filter on the spot and then concentrated for the second time by PEG/NaC1 in 2011. The DNA of concentrated samples were extracted for the quantification of human adenovirus in real-time PCR subsequently to monitor the pollution of human adenovirus in water. Results For the first concentration by NanoCeram filter, the recovery rates were （51.63 ± 26. 60） % in source water and （50. 27 ± 14. 35） % in treated water, respectively. For the second concentration, the highest recovery rate was reached to （90. 09 ± 10. 50 ） % at the concentration of 0. 13 kg/L of PEG 8000. The sequence identity score of standard of adenovirus for real time PCR and adenovirus gene was 99%, implying that it can be successfully used to quantification with human adenovirus. The levels of human adenovirus in the water samples sampled in 2011 ranged from 4. 13 × 103 to 2. 20 × 106 copies/L in source water,while range from 5.57 × 102 to 7.52× 105 copies/L in treated water and the removal efficiency range was （75.49 ±11.71 ）%. Conclusion NanoCeram filers combined with PEG/NaCX was an effective method to concentrate virus in aquatic environment. There was a large number of human adenovirus in source water, and it is not sufficient to remove them thoroughly through conventional water treatment processes.