【作者】 叶晓艳；

【导师】 谷康定；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2012， 博士

【摘要】 由人粪便排入环境中的病毒已达140多种血清型,可导致胃肠炎、肝炎、发热、脑膜炎、心肌炎等疾病,严重威胁到人类健康。这些病毒在胃肠道复制,主要通过粪口途径传播,是导致水源性疾病的一个重要原因。1988年上海30万人暴发甲型肝炎病毒感染,系由于食用遭甲型肝炎病毒污染水体中养殖的毛蚶造成,1991印度的坎普尔由于使用了被戊型肝炎病毒污染的饮用水造成7.9万人感染戊型肝炎。近年来屡见饮水受到肠病毒(enteic virus)污染引起肠道传染病爆发的报道。2007年广东省东莞市一起173例不明原因腹泻流行,经调查证实为诺如病毒污染饮水所致。饮用水安全与人们的生产生活息息相关,其病毒学安全性应该引起学界与有关管理部门高度重视。许多国家都采用粪便污染指示菌,例如粪大肠菌群、总大肠菌群、肠球菌等来评价水体的卫生微生物学质量包括病毒污染状况。研究发现水环境的细菌学水平和病毒浓度之间无明显相关,同时病毒对外界环境和消毒作用的抵抗力更强,在满足水体细菌学卫生标准的情况下仍能检测到病毒。因此目前各国普遍采用的指示菌不能准确反映水中病毒学安全性。直接对病毒进行检测可以提供更可靠的水体病毒污染信息,进一步确保用水安全。但是,迄今国内外尚未制定饮水病毒学标准和统一可行的水中病毒学检测方法,根本原因在于其技术难度高、费用昂贵、工作量大,涉入门槛高,基础科研数据积累少。严重制约了饮用水病毒学研究在国内的开展。因此,本研究将从新材料、新配方、新的分子生物学检测法多角度,探索建立可靠、有效的饮用水中病毒浓缩、检测方法,并在制水生产实践中检验诸方法应用效果。仅就技术而言,首先环境水体中病毒浓度远比临床病人感染样本低,如果特指可能存在于饮用水中的病毒,两者相差最大可达1017数量级。采用最灵敏的临床检测方法也无法对饮水中病毒进行检测。因此,欲开展饮用水病毒学研究,必须面对水中病毒浓缩技术与设备的挑战。目前浓缩水中病毒的方法有：微孔滤膜吸附法、硅藻土吸附法、氢氧化铝吸附沉淀法等。但因为材质和材料物理化学性质、洗脱液性质、操作流程不同,导致各种浓集方法最终结果相差悬殊,使不同国家与地区结果缺乏可比性,难以统一,因此迫切需要探索建立一种高效、灵敏和简捷的浓缩方法。本研究在此方面进行了初步尝试,对比了国产微孔滤膜与进口NanoCeram滤芯初次浓缩病毒的效率。NanoCeram滤芯是一种新型的正电滤芯,因此国外相关实际应用的报道也相对较少。滤芯的表面积大500至600m2/g,能够对不同浊度的大体积水样进行有效浓缩,不需调节水样的pH等处理,可直接进行浓缩,操作过程简单,且价格仅为1MDS滤芯的1/5。本研究首次在国内将此滤芯应用于实际水样病毒的浓缩,所获实验数据为国内今后利用该滤芯提供了良好的参考依据。研究水中病毒的第二个技术难点在于,操作步骤繁杂,第一次浓缩后并不能直接检验病毒,必须再进行第二次浓缩,使其最终体积缩小到类似于临床标本体积。第二次浓缩方法有,絮凝沉淀、透析、超速离心等,由于待浓缩样本体积仍然较大,后两种方法不太实用,主要采用絮凝沉淀法。絮凝剂有无机絮凝剂和有机絮凝剂,种类繁多,理化性质各异,寻找理想满意的絮凝剂,需要具体问题具体分析,开展细致筛选研究。本研究探讨了3种不同絮凝剂在二次浓缩中的最佳条件与效果,有机絮凝剂聚乙二醇择优而出。浓缩过程结束,仅仅类似于完成了临床标本的采集,随之相应的除菌、选择敏感宿主接种样本,观察病毒复制接踵而至。但是,环境中病毒与临床检验巨大不同处是其多样性,一个患者一般情况下只感染一种病毒,其实验室敏感宿主细胞范围较窄,容易确定。可是,环境水体中浓缩的病毒,可能为一种,也可能有多种数十种,不同病毒有其特定的敏感宿主,这为最终检测环境病毒带来极大的工作量。另外,有的病毒不能用细胞检测,例如,诺如病毒,或有些病毒对细胞不敏感,例如,腺病毒F组40、41血清型,这为检测环境病毒带来极大不确定性。若要解决以上问题,只能从分子生物学领域借助工具,基因探针和聚合酶链反应(PCR)似乎能够同时解决以上难题,但是基因探针因其灵敏度太低,已被淘汰,而最新的荧光定量PCR(qPCR)技术视乎可弥补上述所有不足。荧光定量PCR可对模板进行实时监测,精确对起始模板进行定量,且检测时间短,从病毒核酸提取到荧光定量PCR完成仅仅需要4-5小时,荧光定量PCR检测范围宽,灵敏度高,特异性强,显示其在环境病毒学检测中的巨大优势。尽管如此,对于具体的每一种病毒,qPCR技术并非唾手可得,针对不同病毒,其保守区引物设计,内标构建,扩增产物特异性、检测方法的灵敏度等,都需要探索研究。本研究针对不易培养的腺病毒F组40、41血清型、培养难度较大的A群轮状病毒及具有普遍意义的整个肠道病毒属,构建了相应的荧光定量PCR方法,成功实现对自来水厂水样中相应病毒的检测。国外研究显示病毒普遍存在于各种水体,例如江、河、湖、海、地下水,乃至经过净化处理过的自来水都含有一定量的病毒。国内有研究人员检测了渤海中肠道病毒的污染情况,检测发现海水中肠道病毒浓度范围为6.3×107拷贝/L至1.7×106拷贝/L,何等仅仅采集了2L水样,发现轮状病毒在北京水源水中的检出率为34.6%,出厂水中的检出率为11.7%,末梢水中的检出率为22.4%。二十年前,张楚瑜等人对武汉东湖水厂肠道病毒进行检测,原水病毒浓度均值为14.31PFU/L,阳性率为60%,出厂水浓度均值为6.7PFU/L,阳性率为35%,水厂水处理工艺对肠道病毒的去除率为53.18%。当时东湖是武汉武昌区的主要饮用水水源,然而随着东湖水污染的日益加重,最后不得不放弃将其作为饮用水水源,改用长江水为唯一饮用水水源。武汉市是长江与汉江汇聚的大都市,武汉受惠于两江,但也可能遭受其害。原因在于,随着中国经济突飞猛进的发展,长江、汉江环境污染负荷日益加重,有报道,仅2007年一年长江就接受了300多亿吨废水。武汉有关部门最新公布资料,在武汉的18各水源保护地附近就有15处污水排放口,直接威胁到自来水厂取水安全。人们有理由为自己饮水的化学安全性、生物学安全性担心。但是,有关利用长江与汉江作为水源的各自来水厂原水、出厂水是否存在病毒污染,其变化规律如何,一直是有待解开的迷。不仅如此,整个长江流域200多个大中小城市,甚至全国县以上4000多个水厂也几乎很少开展饮水病毒学研究。这种状况与国际上同类研究差距甚远,无论从学术上还是从生产实践中都需要急起直追。本研究对武汉市两江共6个自来水厂病毒污染状况开展了一年四季的监测调查,比较了出厂水与原水中腺病毒、轮状病毒和肠道病毒的污染水平与去除率,考察了粪便污染指示菌、有关重要理化指标与病毒灭活的关系,对研究结果进行了科学谨慎的评价,填补了该领域的部分空白。本研究主要包括下述三个部分：第一部分饮用水中病毒浓缩方法的建立与比较目的：测试评价NanoCeram滤芯对原水和饮用水中病毒初次浓缩的效果,比较无机絮凝Mg(OH)2法、有机絮凝法及PEG/NaCl对初次浓缩液进行二次浓缩的效果。方法初次浓缩：以f2噬菌体为肠病毒代表,将f2噬菌体投加入50L长江、汉江混合原水中,配制模拟原水样本,取100L实验室末梢水,Na2S2O3中和余氯后投加f2噬菌体,配制模拟饮用水样本。检测水样的浊度、温度、pH值,采用NanoCeram正电滤芯浓缩模拟原水和饮用水样中病毒,采用3%牛肉浸膏溶液pH值9.5洗脱滤芯。调整洗脱液pH值为7.0即得初次浓缩液。二次浓缩方法的选择：200ml 3%牛肉浸膏溶液(pH值9.5)中加入一定量f2噬菌体,配制二次浓缩模拟样。比较无机絮凝(Mg(OH)2)法、有机絮凝法及聚乙二醇(PEG)/NaCl法对病毒进行二次浓缩效果差异。无机絮凝法：在模拟水样中加入一定量的1mol/L的氯化镁溶液和1mol/L的磷酸氢二钾溶液,调节溶液pH值为8.4。观察絮凝形成情况,检测样本f2噬菌体的回收率。有机絮凝法：调节二次浓缩模拟水样的pH值为1.5,2.0,3.0,3.5,4.0,5.0,7.0检测不同pH下溶液的浊度及上清和沉淀中f2噬菌体含量,评价其浓缩效果。聚乙二醇(PEG)/NaCl法：比较不同浓度PEG8000的浓缩效果的差异(4%、6%、8%、10%、12%、13%、14%、16%、18%、20%(g/100ml))。f2噬菌体浓度的测定以Ecoil 285为其宿主菌,采用双层琼脂平板法进行检测。结果：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f2噬菌体回收率为50.27%±14.35%。无机絮凝法由于洗脱存在一定困难,回收率较低。有机絮凝法中当溶液pH<3时,溶液浊度较高,但是f2噬菌体的回收率仍较低(<5%)。PEG8000浓度为4%和6%时f2噬菌体回收率小于3%;当浓度达到8%后,平均回收率可达70%以上；浓度达到13%时回收率最高达90.09%士10.50%。结论：采用NanoCeram滤芯进行初次浓缩,联合PEG8000/NaCl(13%)法进行二次浓缩是一种对饮用水病毒进行浓缩的高效方法。第二部分荧光定量PCR检测腺病毒、轮状病毒和肠道病毒方法的建立目的：制备荧光定量PCR检测总的人腺病毒(HAdV)、人腺病毒F(HAdVF)、人轮状病毒A(RVA)和肠道病毒(EV)的标准品,以制备的各病毒重组质粒标准品进行各病毒的绝对定量。方法：采用MA104细胞分离婴儿粪便中人轮状病毒。提取人腺病毒40DNA,脊髓灰质炎病毒1型sabin株和分离的轮状病毒RNA,并逆转录为cDNA。设计合成HAdV、HAdVF、RVA和EV引物,以提取的人腺病毒血清型40 DNA,脊髓灰质炎病毒1型sabin株和分离的轮状病毒cDNA为模板进行梯度PCR优化退火温度,选择最适退火温度进行普通PCR。胶回收纯化各病毒PCR产物,纯化后DNA连接入pMD18-T Vector载体,转化入DH5a感受态细胞,在LB/氨苄/IPTG/X-Gal平板上进行蓝白筛选,LB培养基增殖各阳性克隆菌,提取菌液质粒,测序。测序所得插入片段的碱基序列与模板基因输入NCBI的BLAST序列类似性检索工具进行一致性检测。测定纯化质粒浓度并换算为拷贝数浓度,然后以构建成功的各病毒标准品建立荧光定量PCR检测HAdV、HAdVF、RVA及EV的方法。评价各病毒标准品荧光定量PCR的稳定性和敏感性。反应体系为(20μ1)：模板2μ1,SYBR Green 10μl,上游引物100nm/μ1,下游引物100nm/μl,无菌水补至20μl。反应条件为：50℃2min;95℃10min循环40次95℃15s,60℃15s,72℃30s,融解曲线：65℃-95℃。采用构建的标准品定量不同稀释度的人腺病毒血清型40,脊髓灰质炎病毒1型(Sabin株)及人轮状病毒A,并比较各稀释度病毒滴度与拷贝数浓度的相关性。结果：HAdV、HAdVF、RVA和EV普通PCR最佳退火温度为60℃,蓝白筛选显示连接、转化成功,测序分析显示各病毒的重组质粒中含有各病毒的目的片段。所构建的HAdV荧光定量PCR标准品与目的基因片段(132 bp)一致度为99%；HAdVF标准品与目的基因片段(130 bp)一致度为100%；RVA标准品与目的基因片段(161bp)一致度为100%；EV标准品与目的基因片段(143bp)一致度为99%；各病毒荧光定量PCR标准品构建成功,可用于各病毒的绝对定量。纯化后质粒浓度分别为1.1×1011拷贝/μ1(HAdV);3.9×1010拷贝／μ1(HAdVF)；9.3×1010拷贝/μ1(EV)6.1×1010拷贝(?)μl(RVA)。不同稀释度HAdV, HAdVF, RVA和EV(10-108拷贝/反应),扩增曲线分明,融解曲线呈单峰,且Tm分别为：85.4℃(HAdV),82.6℃(HAdVF),74.8℃(RVA),83.4 (EV),扩增效率为89%-99%。各病毒荧光定量PCR反应灵敏度为10拷贝／反应,HAdV稀释度对数值与拷贝数浓度对数值存在线性相关,且R2=0.905。RVA稀释度对数值与拷贝数浓度对数值存在线性相关,且R2=0.986。HAdVF稀释度对数值与拷贝数浓度对数值存在线性相关,且R2=0.998。脊髓灰质炎病毒稀释度对数值与拷贝数浓度对数值存在线性相关,且R2=0.967。结论：利用制备的HAdV、HAdVF、EV、RVA的重组质粒标准品进行荧光定量PCR获得的标准曲线具有较高的扩增效率和良好的线性关系,且产物特异性好,具有良好的重复性和稳定性,该方法可用于对HAdV、HAdVF、EV和RVA的检测。本研究建立的实时荧光定量PCR检测HAdV、HAdVF、EV和RVA的线性范围为10-1.0×108拷贝/μ1,检测的灵敏度可达10拷贝／反应体系,能够用于低浓度样本的检测。荧光定量PCR检测HAdV、HAdVF、EV和RVA所得拷贝数值与稀释度之间存在高度的相关性,对病毒的定量准确,可靠。第三部分水厂原水和出厂水中腺病毒、轮状病毒、肠道病毒的测定目的：利用第一部分建立的饮用水中病毒浓缩方法和第二部分建立的HAdV、HAdVF、RVA和EV荧光定量PCR的方法,检测武汉市6个水厂2011年4个季节原水和出厂水中HAdV, HAdVF, RVA和EV的污染情况,并对水厂的处理效果进行评价,探索病毒浓度和其他检测指标的相关性。方法：于2011年1月、4月、7月和10月用NanoCeram滤芯现场浓缩武汉市六家自来水厂(其中三座水厂以汉江水为水源水,另外三座以长江水为水源水)原水100L和饮用水200L,同时测定原水中pH、温度、浊度、粪大肠菌群和出厂水中pH、温度、浊度、游离余氯及粪大肠菌群。饮用水用终浓度为50mg/1 Na2S2O3中和余氯。每个样本更换一个滤芯,将滤芯置于冰盒中低温运输至实验室,3%牛肉浸膏溶液(pH值为9.5)600m1进行洗脱。调整洗脱液pH值为7.0,采用聚乙二醇(PEG)/NaCl法进行二次浓缩,于洗脱液中加入PEG8000(终浓度为13g/100ml),NaCl(1.2g/100ml),完全溶解后4℃静置3h,10000g,离心30min,沉淀用PBS(pH值为7.0)洗脱,终体积为10ml。提取浓缩液中病毒DNA和RNA,并将RNA逆转录为cDNA,进行荧光定量PCR检测,监测原水和饮水中HAdV、HAdVF. RVA和EV污染状况,同时评价水厂的水处理效果,探索各病毒浓度、粪大肠菌群及浊度之间的相关性。评价原水和出厂水中可能产生的PCR抑制效应,及离心和稀释处理对降低该效应的作用。结果：2011年,武汉市六个自来水厂水源水和饮用水中HAdV. HAdVF和RVA的阳性率均为100%,EV原水中的阳性率为46%,饮用水中的阳性率为21%。长江水原水中各病毒浓度范围如下HAdV:5.54x103拷贝/L-2.20x106拷贝/L; HAdVF:5.69x104拷贝/L-3.76×105拷贝/L；RVA：6.91×103拷贝/L-1.11×105拷贝/L；EV：0～7.95×103拷贝/L。汉江水原水中各病毒浓度范围为HAdV:2.75x103拷贝/L-1.05×1O6拷贝/L；HAdVF:5.66×102拷贝/L-4.81×104拷贝/L;EV：0-5.56×103拷贝/L；RVA:4.84x103拷贝/L-4.52×104拷贝/L。EV清除率为97%,RVA为82%,HAdV为73%,HAdVF为72%。PCR抑制实验中冬季原水投加一定量的HAdV标准品,检出百分比为57.04%,春季原水检出百分比为61.20%,冬季出厂水为96.46%,春季出厂水为91.07%,原水中PCR抑制作用较严重。比较同一样本离心前后HAdV浓度变化发现,对于不同样本离心前后HAdV浓度在不同程度上均有所增加,增加百分比为29.7%-198.2%(t=10.235,P=0.000)。而同一样本离心前后RVA浓度变化不明显(t=1.027,P=0.338)。各样本DNA稀释两倍后稀释液加标回收率较原液加标回收率均有不同程度的增加,增加回收率为8.70%～55.90%。RVA cDNA稀释液加标回收率较原液加标回收率均有不同程度的增加,增加回收率为6.43%-37.87%。长江原水HAdV浓度与浊度之间存在一定的正相关,(R2=0.768,P=0.004),汉江原水HAdVF与浊度之间存在一定的正相关相关,(R2=0.711,P=0.010)。其他病毒浓度与浊度之间的相关性并未发现。粪大肠菌群浓度与各病毒浓度之间均不存在明显的相关性。RVA长江水和汉江水含量有差异,长江中浓度高于汉江(P=0.035)。结论：离心和稀释预处理是环境样本中去除PCR抑制物的有效方法。本研究建立的病毒浓缩方法和荧光定量PCR研究结果表明,长江和汉江水原水中存在一定量的HAdV、HAdVF、RVA和EV。在满足生活饮用水卫生标准的出厂水中仍能检测出HAdV、HAdVF、RVA和EV,现有的水处理措施尚不能保证对病毒的完全去除。更多还原

【Abstract】 More than 140 types of enteric viruses are found in aquatic environments which can cause a wide variety of illnesses in humans such as hepatitis, gastroenteritis, meningitis, fever, rash, conjunctivitis and even diabetes. These viruses are transmitted via the fecal oral route and primarily infect and replicate in the gastrointestinal tract of the host. They are major causes of waterborne and water-related diseases. Extreme examples were the outbreak of 300,000 cases of hepatitis A and 25,000 cases of viral gastroenteritis caused by shellfish harvested from a sewage-polluted estuary in 1988 in Shanghai. In 1991, an outbreak of 79,000 cases of hepatitis E in Kanpur was ascribed to polluted drinking water. There were 173 cases diarrhea reported to be caused by drinking water polluted by norovirus in Dongguan in Guangdong province in 2007. Therfore, more attention should be paid to the viral safety of drinking water by both governments and public because of the effect of viruses on human health.In many countries, including the United States, bacterial indicators such as fecal coliform total coliform and enterococci are adopted to assess the microbiological quality of water. But no significant correlation was shown between levels of bacterial indicators and concentrations of enteric viruses. There were still some enteric viruses detected in drinking water which had met the bacteriological standards. So it is unsafe to completely rely on bacteriological standards to assess the viral quality of any kind of water. To detect the enteric viruses directly can supply more reliable information about viral contamination of water. However the standards of permitted virus number in water and detection method for viruses have not be made and standardized so far because of the difficulty in detection, high cost and workload. The concentration of virus in aquatic environments is usually much lower than that from clinical specimen, and the concentration of a certain virus in clinical specimen could be as 1016 times high as in drinking water. It is difficult to detect the virus in drinking water directly even if using the most sensitive method. Thus the most important precondition is to concentrate the virus from water.The common methods for virus concentration are microporous membrane, diatomaceous filters and glass wool filters. The efficiency varies with different methods. It is necessary to explore an effective, sensitive and convenient method for viral concentrateion. The recovery efficiency of microporous membrane and NanoCeram filter were compared in this study. NanoCeram cartridges, a new electropositive filter with a surface area of 500 to 600 m2/g, can concentrate viruses in water with high turbidity. It is convenient to use the cartridge and the price of it is only 1/5 of 1MDS filter. It is the first time that NanoCeram filter has been used to concentrate viruses in water samples in China.The second concentration of viruses in eluates from washed cartridges is needed because it is still difficult to detect viruses directly after first concentration. Coagulation-precipitation, dialysis and ultracentrifugation are commonly applied for the second concentration. Dialysis and ultracentrifugation are not useful for large volume of first concentrated solution. Flocculants include organic and inorganic chemicals. Three kinds of flocculants were compared in this study and PEG process had the highest efficiency in second concentration among them.After second concentration of the viruses, the volume of the sample solution reduces to small volume similar to clinical specimens which are easy to be assayed with common methods. However, one of huge differences between the environmental and clinical samples for virus test is its diversity. A clinical patient is generally infected with one kind of virus that is readily diagnosed in a laboratory. But for environmental samples, there may be one or much more kinds of viruses in one concentrated sample. Because the susceptibilities of different viruses to their cellular hosts are various, a huge amount of work is required to identify each of them. In addition, there were some kinds of viruses such as norovirus which cannot propagate in cell culture and other kinds of viruses, such as adenovirus serotype 40 and 41 which are difficult to multiply in cells. Molecular biological techniques like gene probe and PCR can overcome these shortages. Although the sensitivity of gene probe is too low to be used, the state of real time PCR has shown many advantages, for example, it is inexpensive, timesaving, and able to detect nonculturable viruses or slow-growing viruses. It is very useful to estimate the public health risks of low levels of enteric viruses in aquatic environments. To perform qPCR, the nucleic acid standards for real time PCR shoud be constructed separately according to different individual virus. The stability and sensitivity of qPCR reaction system should be evaluated. In this study adeno virus serotype 40 and 41, rotavirus group A which are difficult to culture and enterovirus in water samples, were detected by real time PCR Enteric virus were found in many kinds of aquatic environments such as river, lake, sea underground water and even treated drinking water.Chinese investigator detected the enteroviruses in Bohai bay in Tianjing, with the concentration from 6.3×107copies/L tol.7×106copies/L. Rotaviruses were also detected in surface water, raw water, treated drinking water and tap water, with positive rates of 34.6%,11.7%,22.4% respectively in only 2 liters water using RT-nested PCR in China. Twenty years ago, Zhang investigated the presence of enteric viruses in source water of the East Lake and tap water of drinking water treatment plants using cell culture assay in Wuhan. They found that 60% of the raw water samples and 35% of tap water samples were positive for enteric viruses respectively. The average viral removal rate of drinking water treatment plants was only 53.18%. The East Lake as a major drinking water source at that time was polluted severely year by year and was finally abandoned as a potable water resource in the middle of the 1990s. Instead, river water has been the only water resource for drinking water production in the city. However, little has been known about the status of virus pollution in source water and tap water coming from rivers in Wuhan, or in other cities on rivers in China since then.With China’s rapid economic development, the Yangtze River and the Hanjiang River have been increasingly polluted by the discharge of untreated or incompletely treated industrial waste, agricultural waste and sewage. By 2007, yearly industrial and urban wastewater released into the Yangtze had exceeded over 30 billion tons,50% of which was untreated. It was reported that there are 14 sewage outfalls near the 18 source-water protection areas, which threatened the quality of source water. The situation of virus in the Yangtze River and the Hangjiang River is still unknown. What is more, little information on viral pollution in any drinking water treatemant plant using both River water as resourses has been reported so far. For the present report, source water and finished water from six water works depending on the Yangtze and the Hanjiang Rivers were collected in Wuhan in four different seasons. Adenovirus, rotavirus and enterovirus were assayed by real time PCR to examine the viral status in the water as well as some other aspects that could impact the real time PCR measurement.Part I Concentration of Virus in Drinking WaterObjective To test and assess the concentration recovery efficiency of viruses with NanoCeram filters, and compare the recovery efficiency of inorganic flocculation with Mg(OH)2, organics flocculation and Polyethylene Glycol (PEG)/NaCl precipitation for the second concentration.Methods For the first concentration:f2 bacteriophage, standing for the enteric virus in water, was spiked to 50L of the source water from the Yangtze River and the Hanjiang River to simulate source water and was spiked to 100L tap water dechlorinated by Na2S2O3 to simulate treated drinking water. The simulated waters were filtered through NanoCeram filters, and then the filters were eluted with 3% beef extract (pH 9.5). After that, the pH of elution solutions were adjusted to 7.0. For the second concentration, f2 phage was spiked into 200ml of 3% beef extract (pH 7.0) to simulate the first concentrated solution. The recovery efficiency of virus by inorganic, organic flocculation and PEG/NaCl precipitation were compared for second concentration. For inorganic flocculant, MgCl2(1M) and K2HPO4(1M) were added to the simulated water and then the pH was adjusted to 8.4 and the recovered precipitate was quantitated subsequently for f2 phage. In the organics flocculantion, pH of the simulatied water was adjusted to 1.5,2.0,3.0,3.5,4.0,5.0, 7.0, and the turbidity of each pH was tested, together with the concentration of f2 phage. The recovery efficiency was compared in different conditions. In PEG/ NaCl system, the recovery efficiency of f2 phage in different concentrations of PEG 8000(4%、6％、8%、10%、12%、13%、14%、16%、18%、20%) was compared. A double agar-layer technique was applied to calculate the concentration of f2 bacteriophage using E coli 285 as its host bacteria.Result The average recovery yield was 50.27%±14.35% for tap water samples and 51.63%±26.60% for source water samples using NanoCeram cartridges for the first concentration. The recovery yield was very low by using inorganic flocculants. Although the turbidity of solution (an indicator of forming flocculate) was getting higher when the pH value was below 3, the recovery yield was still very low(less than 5%) in organic flocculantion. When the concentrations of PEG 8000 were 4% and 6%, the recovery yield was less than 3%, while the concentration reached 8%,the average recovery yield was larger than 70%.The recovery yield was largest (90.09%±10.50%) at the concentration of PEG 8000 being 13%.Conclusion NanoCeram filers combined with PEG/NaCl is an effect method to concentrate virus in drinking water.Part II Construction of Standards for Adenovirus, Rotavirus and Enterovirus for Real Time PCRObjective To constructs nucleic acid standards for HAdV, HAdVF, EV and RVA for real time PCR which were used to quantitate the concentration of each virus.Method The RNA of poliovirus 1 Sabin type and human rotavirus separated from infant feces was extracted using High Pure Viral Nucleic Acid Kit (Roche, Germany), and then reverse-transcribed to cDNA. DNA of adenovirus type 40 was also extracted. The conditions of conventional PCR were optimized with the genomic DNA and cDNA of different viruses by gradient PCR. The templates of each virus were subsequently amplified using the appropriate primers of different viruses in conventional PCR protocol. DNA fragments were recovered from agarose gels, and then were cloned into a pMD 18-Tvector using the TA cloning strategy. The plasmids were transformed into E.coli DH5a and recombinant bacteria were selected on ampicillin-containing LB agar. The positive clones were propagated overnight in liquid culture. The plasmids were extracted from E. coli and the inserts were sequenced to confirm their specificity. Then the sequences were compared to the data posted in GenBank by using the BLAST algorithm. DNA concentration was measured and the copy number was calculated according to the formula. Real time PCR was carried out with the standards of each virus to quantitate the concentration of HAdV, HAdVF, RVA and EV. The stability and sensitivity were assessed. Each real-time PCR mixture (20μl) contained 10μl 2×PCR mix,100 nM each primer, and 2μl DNA template. The amplification procedures included three hold programs, (ⅰ) 2 min. at 50℃, (ⅱ) 10 min. at 95℃, followed by 40 cycles consisting of 15 seconds at 95℃,30seconds at 60℃and 30seconds at 72℃, (ⅲ) then a dissociation curve analysis from 60℃to 95℃. The concentrations of different diluted virus of adenovirus, poliovirus and rotavirus were quantitated by the constructed real time PCR. Meanwhile the relationship between the copy number and dilutions with eachvirus were assessed.Result The best annealing temperature of HAdV, HAdVF, RVA and EV was 60℃in conventional PCR reaction. The courses of connection of specific DNA fragments to pMD 18 plasmid and transformation of the recombinant plasmid to susceptible bacterial cells were successful. The recombinant plasmid included target products. The identities of inserted nucleotide sequences of HAdV, HAdVF, RVA and EV to their general template sequences were 99%(132bp),100%(130bp),100%(161bp), and 99%(143bp), respectively. The concentrations of the final plasmid were 1.2×1011copied/μl (HAdV),3.9x1010copied/μl (HAdVF),9.3×1010copied/μl (EV), and 6.1×010copied/μl (RVA). The amplifying curves with different concentrations of the standards of HAdV, HAdVF, RVA and EV from 10 to 108 copies/μl were separated well and there were only one peak in dissociation curve with each virus with the Tm value of 85.4℃(HAdV),82.6℃(HAdVF),74.8℃(RVA),83.4(EV). The amplifying efficiency were ranged from 89% to 99%.The linear correlations between the copy numbers and titers with different viruses were as follow:R=0.905 for HAdV, R2=0.986 for RVA, R2=0.998 for HAdVF, R2=0.967 for EV.Conclusion The construction of external standards for detection of HAdV, HAdVF, EV and RVA in water using real-time PCR was successful. The established qPCR assay based on recombinant plasmid as standards has good reliability,, repeatability and sensitivity to quantify HAdV, HAdVF, EV and RVA in water. As few as 10 copies can be reliably detected using the standards of HAdV, HAdVF, EV and RVA by real-time PCR. There are close linear correlation between the copy numbers and titers for each virus.PartⅢDetection of Adenovirus, Rota virus and Entero virus in Source Water and Treated Drinking WaterObjective To quantify the concentration of total human adenovirus(HAdV), human adenovirus group F(HAdVF), human rotavirus group A(RVA) and enterovirus(EV) in source water and treated drinking water in four seasons in 2011, by using the constructed method of concentration in part 1 and the method of real time PCR in part 2. To evaluate the viral removal efficiency of water treatment processes in drinking water treatment plants and its correlation with different physicochemical and biological parameters.Method Water samples(1001 for source water,2001 for finished water) were collected from the six drinking water treatment plants (three drinking water treatment plants are situated on the banks of the Yangtze River, and the other three plants on the banks of the Hanjiang River) in four seasons separately:winter (Jan.2011); spring (Apr.2011); summer (Jul.2011) and autumn (Oct.2011). In order to obtain more information about the water characteristics, several physicochemical parameters such as turbidity, temperature, pH, and residual chlorine as well as fecal coliform were measured. Finished water (2001) dechlorinated by adding 50 mg of sodium thiosulfate per liter and source water (1001) were filtered through NanoCeram VS2.5-5 virus filters. Filters were changed for each sample and used only once. The cartridges of the samples were transported to the laboratory in ice box after collection and were subsequently eluted by 600m1 3% beef extract (pH 9.5). PEG/NaCl precipitation was used for the second concentration. PEG8000 (13%) and NaCl (1.2%) were added to the elution solution adjusted the pH to 7.0. Then the solution was centrifugated at 10000g for 30min. The pellet was finally suspended in 10 ml PBS (pH 7.0). The nucleic acids of water samples were extracted. RNA was reverse-transcribed for cDNA synthesis. Real-time PCR was performed by SYBR Green I fluorescent dye strategy to quantify the concentration of HAdV, HAdVF, RVA and EV in water samples. The viral removal efficiency of water treatment process in drinking water treatment plants was calculated and the correlations with different parameters were analyzed by Pearson correation. The PCR inhibition effects in source water and treated water were comparably evaluated by adding a given amount of standard nucleic acids and two ways, centrifugation and dilution, of removal of those PCR inhibitors were adopted.Result HAdV, HAdVF and RVA were all positively detected in the samples of source water and treated drinking water. EV could be found in 46%(11/24) of all the source water samples, but only 21%(5/24) positive in treated drinking water. The concentration of enteric viruses in source water of the Yangtze River were as follow: 5.54×103copies/L～2.20x 106copies/L (HAdV); 5.69×104copies/L-3.76×105copies/L(HAdVF) ;6.91×103copies/L～1.11×105copies/L (RVA); 0～7.95×103copies/L(EV). The concentration of enteric viruses in source water of the Hanjiang River were as follow 2.75×103copies/L～1.05x106copies/L(HAdV); 5.66×102copies/L～4.81×104copies/L(HAdVF); 0～5.56×103copies/L(EV); 4.84×103copies/L～4.52×104copies/L(RVA). The highest removal rate was EV (97%), followed by RVA (82%), HAdV (73%) and HAdVF (72%). In our study, about 57.04% and 61.20% of the spiked DNA could be detected in the nucleic acids of source water samples, while 96.46% and 91.07% of them could be detected in treated drinking water, indicating that there were some factors influencing the underestimating of the viral concentrations of water samples. The increased rates of recovery via seeding HAdV standards ranged from 29.7% to 198.2% in various sets of nucleic acid extraction (t=10.235, P=0.000). However no improvement was observed for RVA in source water samples after centrifugation (t=1.027, P=0.338). There were significant correlations between the concentration of HAdV and the turbidity of the Yangtze River (R2=0.768, p=0.004), between HAdVF and the turbidity of the Hanjiang (R2=0.711, P=0.010). No correlation between fecal-indicator bacteria and enteric viruses was observed. The concentration of rotavirus in the Yangtze River was higher than that in the Hanjiang River (P =0.035).Conclusion Although the parameters of pH, turbidity, fecal coliforms and residual chlorine have all met the national standards for drinking water, a large number of HAdV, HAdVF, RVA and EV in source water and finished water have been measured by qPCR in six water works in Wuhan. Centrifugation and dilution are good ways to get rid of PCR inhibitor to increase the accuracy of qPCR assays.更多还原