三氯化铁-聚偏氟乙烯膜滤器富集噬菌体方法的建立和评价  

Establishment and evaluation of a method for phages enrichment by ferric trichloride-polyvinylidene fluoride membrane filter

作　　者：张华尧 田哲[2] 唐宋 周海健[4] 樊粉霞[5] 阚飙 Zhang Huayao;Tian Zhe;Tang Song;Zhou Haijian;Fan Fenxia;Kan Biao(Department of Epidemiology and Health Statistics,School of Public Health,Shandong University,Jinan 250012,China;Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences/State Key Laboratory of Environmental Aquatic Chemistry,Beijing 100085,China;National Institute of Environmental Health,Chinese Center for Disease Control and Prevention/China CDC Key Laboratory of Environment and Population Health,Beijing 100021,China;National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Disease Prevention and Control,Beijing 102206,China;Department of Diarrheal Diseases,National Institute for Communicable Disease Control and Prevention,Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Disease Prevention and Control,Beijing 102206,China)

机构地区：[1]山东大学公共卫生学院,济南250012 [2]中国科学院生态环境研究中心环境水质学国家重点实验室,北京100085 [3]中国疾病预防控制中心环境与健康相关产品安全所、中国疾病预防控制中心环境与人群健康重点实验室,北京100021 [4]中国疾病预防控制中心传染病预防控制所、传染病预防控制国家重点实验室,北京102206 [5]中国疾病预防控制中心传染病预防控制所腹泻病室传染病预防控制国家重点实验室,北京102206

出　　处：《中华预防医学杂志》2023年第3期443-450,共8页Chinese Journal of Preventive Medicine

基　　金：国家重点研发计划(2021YFC2300304)。

摘　　要：目的建立和评价基于三氯化铁-聚偏氟乙烯(FeCl_(3)-PVDF)膜滤器富集自然水体中噬菌体的方法。方法基于絮凝浓缩原理,利用铁离子絮凝结合膜过滤器建立从水样中回收噬菌体的方法。利用琼脂双层法测定噬菌体滴度、噬菌体荧光染色观察和实时荧光PCR反应等方法检测噬菌体的回收效率。采集不同来源的水体标本进行模拟实验评价富集效果,同时以医院排出污水作为实际水样,利用临床常见的耐药菌作为宿主指示菌,进一步分析FeCl_(3)-PVDF膜滤器富集方法对自然实际水样中噬菌体的富集效果。结果建立了铁离子浓度50 mg/L,采用PVDF膜滤器富集自然水体中噬菌体的方法。该方法对噬菌体的回收率为93%~100%;在多功能显微镜下观察发现富集后水样的噬菌体明显增多,酶标仪测定富集后水样荧光值约为富集前的13倍;对医院外排水实际水样浓缩处理后,浓缩组和未浓缩组噬菌体的分离阳性率分别为23%和4%,浓缩组荧光值为未浓缩组的2~24倍。结论FeCl_(3)-PVDF膜滤器方法可简便、高效快速地富集不同水体样本中噬菌体。Objective To establish and evaluate a method of enriching bacteriophages in natural water based on ferric trichloride-polyvinylidene fluoride(FeCl_(3)-PVDF)membrane filter.Methods Based on the principle of flocculation concentration,the method of recovering bacteriophage from water sample was established by using iron ion flocculation combined with membrane filter.The titer of phage was determined by Agar double layer method.The recovery efficiency of phage was detected by phage fluorescence staining and real-time fluorescence PCR reaction.Water samples from different sources were collected for simulation experiment to evaluate the enrichment effect.At the same time,the sewage discharged from hospitals was taken as the actual water sample,and the common clinical drug-resistant bacteria were used as the host indicator bacteria to further analyze the enrichment effect of FeCl_(3)-PVDF membrane filter rapid enrichment method on the bacteriophage in natural water samples.Results The method of enrichment of bacteriophages in natural water by iron ion concentration 50 mg/L and PVDF membrane filter was established.The recovery rate of this method for bacteriophage was 93%-100%.Under the multi-functional microscope,it was found that the bacteriophage of the enriched water sample increased significantly and the fluorescence value of the enriched water sample determined by the enzyme labeling instrument was about 13 times as high as that before enrichment.After concentration of the actual water samples from the hospital drainage,the positive rate of bacteriophage isolation in the concentrated group and the non-concentrated group was 23%and 4%,and the fluorescence value in the concentrated group was 2-24 times as high as that of the non-concentrated group.Conclusion The method of FeCl_(3)-PVDF membrane filter is a simple,efficient and rapid method for enriching bacteriophages in different water samples.

关 键 词：噬菌体 富集 膜滤器 回收率 

分 类 号：R12[医药卫生—环境卫生学]