环境水体中活性腺病毒有效检测体系的构建及在水质监测中的初步应用

发布时间：2018-07-16 09:24

【摘要】：背景:当前,娱乐休闲水质的评估标准主要基于对粪便污染指示菌(fecal indicator bacteria,FIB)的检测,然而,这些标准难以准确预测人类水源相关致病性病毒的存在。在过去二十年中,因娱乐休闲水污染引起的肠道疾病在逐年增加。美国环境保护署(EPA)提出,需寻找新的可替换的指示物作为监测水质的标准。研究表明人类腺病毒(HAdV)可作为粪便污染的较好候选指示微生物[l],因为在水环境中HAdV相较其他肠道病毒更加稳定,此外,HAdV也被指出与许多娱乐休闲水相关疾病的暴发有关。由于环境水中]HAdV的浓度及回收效率偏低,敏感快捷的PCR检测又无法鉴别病毒的感染活性,故检测水环境中具有感染活性的腺病毒是目前亟待解决的难题之一。为了克服这一缺陷并探索用腺病毒作为指示物对水质的监测的可能性,本研究以HAdV作为模型,测试并建立从环境水中有效浓缩回收有感染活性腺病毒的新方法,评估了腺病毒在海洋贝类中的富集情况。此外,本研究还尝试建立有感染力的病毒颗粒与病毒DNA拷贝数的关联。方法与结果：初步的研究结果显示,腺病毒空斑形成试验的优化条件为：接种0.8x105/ml的A549细胞于孔板,在PH为6.4-7.6的情况下,感染：HAdV孵育约1.5小时-2小时,最后每孔加入1.0毫升1.6%的DMEM-琼脂糖覆盖,培养感染HAdV的细胞约9-10天待空斑形成,以结晶紫-福尔马林进行固定及染色。该优化的空斑技术条件应用于整个研究中,以获得稳定有具有统计学意义的数据结果。为了有效浓缩环境水中的病毒,我们比较了氯化铝预处理或无处理的负电荷滤膜、五种洗脱缓冲液和浓度、优化了洗脱方式和时间,回收的病毒通过空斑形成数确定。结果表明,氯化铝预处理HA负电荷滤膜后,以1.0mM氢氧化钠溶液磁力搅拌洗脱30分钟能回收浓缩约80%的腺病毒。而三种从贝类组织中浓缩HAdV的方法中,3%牛肉提取物洗脱-沉淀法可以浓缩回收约90%的腺病毒。另外,为了评估腺病毒在水环境的稳定性,等量的HAdV分别接种于海水、水管水、PBS及废水中。结果显示,HAdV在海水中较不稳定,且在纯废水中稳定度较高。我们还监测了HAdV在海洋软体生物中的蓄积情况,通过从环境水中采集的贝类养殖在实验室水族缸内,并接种一定量腺病毒,在不同的养殖时间,取水和贝类,利用空斑形成试验及实时荧光定量PCR数据观察腺病毒在海水及软体生物内的浓度变化。研究结果表明,HAdV可蓄积在贝类胃上皮,并在第5天达到最高。这一结果支持了预计的假设：贝类可作为生物指示物以监测水质。结论：本研究通过一系列实验与检测从而建立了从不同水体环境中浓缩腺病毒的优化方法,并评估了腺病毒在不同水体环境中的稳定性及蓄积情况,这对于使用这一病毒作为指示物是极其重要的意义,同时我们还证实了,海洋贝类可作为生物指示物监测水质的可能。综上所述,这一有效敏感的浓缩和检测HAdV方法的建立,为今后利用人类腺病毒作为可替代的补充指示物加强对娱乐休闲水源污染的监测和健康风险评估的可行性奠定了坚实基础。
[Abstract]:Background: the current assessment criteria for recreational and recreational water quality are mainly based on the detection of fecal indicator bacteria (FIB). However, these standards are difficult to accurately predict the existence of human source related pathogenic viruses. In the past twenty years, intestinal diseases caused by recreational water pollution have increased year by year. The EPA proposed to find new replaceable indicators as a standard for monitoring water quality. Studies have shown that human adenovirus (HAdV) can serve as a preferable candidate indicator for fecal contamination, [l], because HAdV is more stable than other enterovirus in water environment, and HAdV is also pointed out with many recreational and recreational water related diseases. Due to the low concentration and recovery efficiency of]HAdV in environmental water, sensitive and quick PCR detection can not identify the virus infection activity, so it is one of the difficult problems to be solved to detect the infected adenovirus in water environment. In order to overcome this defect and explore the monitoring of water quality by adenovirus as a indicator In this study, we use HAdV as a model to test and establish a new method for effective concentration and recovery of infected activated adenovirus from environmental water, and to evaluate the enrichment of adenovirus in marine shellfish. In addition, this study also attempts to establish the association of infected virus particles with the number of DNA copies of the virus.
Methods and results: the preliminary results showed that the optimization conditions for the adenovirus plaque formation test were: inoculated 0.8x105/ml A549 cells on the orifice and PH 6.4-7.6, the infection: HAdV was incubated for about 1.5 hours -2 hours, and 1 ml of DMEM- agar was added to each hole, and the cells infected with HAdV were to be empty for about 9-10 days. The plaque is formed and fixed and dyed with crystal violet formalin. The optimized space spot technical conditions are applied to the whole study to obtain stable and statistically significant data.
In order to effectively concentrate the virus in the environmental water, we compared the aluminum chloride pretreated or untreated negative charge filter, five kinds of elution buffer and concentration, optimized the elution mode and time. The recovered virus was determined by the number of plaque formation. The results showed that aluminum chloride was pretreated with HA negative charge filter and 1.0mM sodium hydroxide solution magnetic stirring. 30 minutes of elution can recover about 80% of the adenoviruses concentrated. And three kinds of beef extract elution - precipitation method can concentrate about 90% of the adenoviruses from the three methods of concentrating HAdV from shellfish tissues.
In addition, in order to evaluate the stability of adenovirus in water environment, the equal amount of HAdV was inoculated in seawater, water pipe, PBS and wastewater. The results showed that HAdV was more unstable in the sea water and had higher stability in pure waste water. We also monitored the accumulation of HAdV in marine mollusks, and by the shellfish culture collected from the environmental water. Laboratory aquarium, inoculated with a certain amount of adenovirus, in different breeding time, water and shellfish, using plaque formation test and real-time fluorescence quantitative PCR data to observe the changes in the concentration of adenovirus in seawater and mollusc. The results show that HAdV can accumulate in the shellfish gastric epithelium and reach the highest in fifth days. This result supports the results. The expected hypothesis is that shellfish can be used as biological indicators to monitor water quality.
Conclusion: in this study, a series of experiments and tests have been made to establish the optimization of adenovirus from different water environment and evaluate the stability and accumulation of adenovirus in different water environment. It is very important for the use of the virus as an indicator. The possibility of monitoring water quality as a biological indicator. To sum up, this effective and sensitive enrichment and detection of HAdV method has laid a solid foundation for the future use of human adenovirus as an alternative complementary indicator to strengthen the monitoring of recreational and recreational water pollution and the feasibility of health risk assessment.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R123.1

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