水体病毒浓缩方法的建立和优化研究

采用氯化钙(CaC l2)、聚乙二醇(PEG,pH7.0)、聚乙二醇(PEG,pH11.5)、三氯化铝(A lC l3)沉淀、Am icon Utcra超滤离心装置和硝酸纤维素吸附膜6种浓缩方法,浓缩人工添加于水体的1型脊髓灰质炎疫苗病毒(PV1),并对浓缩实验条件进行选择和优化。结果表明,CaC l2和聚乙二醇(pH7.0)沉淀法适用于浓缩大容量水体中的病毒,而超滤离心管浓缩法适用于小容量水体,这3种浓缩方法的病毒回收率均达到100%。     

三氯化铝沉淀法浓缩水体中的病毒

近年来, 水源性病毒疾病的爆发在世界范围内被大量报道, 大量研究证实^[1], 外环境水体及淤泥是病毒存活循环的主要场所, 并因此频频引发传染病的流行。但在生活环境的各种水体中, 除生活污水外, 其所含有的病毒浓度往往很低, 以目前现有的检测方法, 仍然达不到不经浓缩而直接从水中检测病毒的目的。同时,食源性病毒的感染剂量非常低, 10?100 个病毒粒子即可引发感染。病毒在离体条件下存活力很强, 对各种理化因子有较强的抵抗力, 耐乙醚和弱酸, 用氯仿、反复冻融、超声波处理都不能使其失活。因此, 水环境中微量存在的病毒仍然对人类的健康带来很大的威胁。在我国, 虽然已经建立起了以PCR 为主导的病毒快速检测方法, 但是对于水样本的前处理技术至今没有找到十分理想的病毒浓缩方法, 无法有效去除样本中的抑制物, 这些因素都严重制约了针对水中食源性病毒的检测和预防。要掌握各种水体中病毒污染的基本状况, 样品中病毒的浓缩是能否得以成功检测的关键。另外, 目前世界上绝大多数国家所执行的标准中, 对病毒的检测标准及其指示生物都没有做出明确说明,归根结底的原因是由于至今没有建立起有效的方法。有鉴于此, 针对水中病毒研究的热点和存在问题, 本刊2009 年第1 期发表了寇晓霞、吴清平等^[2]针对自来水和污水两种不同水体中病毒的浓缩方法。在现有的检测和浓缩方法的基础上, 通过不断创新, 解决现有方法中存在的主要问题, 摸索制约水体中病毒浓缩方法的关键点, 评价不同方法的优劣和实用性。三氯化铝沉淀法最大的优点是可适用于绝大多数具有不同水质特性的水样, 特别是可以克服膜过滤法因易堵塞而对水体悬浮物浓度有严格限制的缺陷。另外, 就检测条件而言, 该方法无需过滤装置等, 也省去进口的阳电滤膜, 从应用推广的角度更为方便, 费用成本低, 对于系统研究我国水中常见食源性病毒检测和监测有一定的应用价值。目前, 该课题组对水体中病毒浓缩方法进行了多方面的应用。对广州河涌水的病毒污染情况进行了调研,初步了解和掌握了病毒的污染状况。广州市河涌水中食源性病毒污染的总阳性率为37.8％。其中有7 个点呈诺如病毒阳性, 8 个点呈轮状病毒阳性。文献[3?4]报道此类病毒的流行月份、季节性与环境水中调查的病毒污染情况基本吻合, 说明了临床发病与环境中此类病毒的污染有一定的相关性。另外还有些点呈甲肝病毒阳性和星状病毒阳性。调研结果表明三氯化铝沉淀法浓缩水体中病毒的方法具有实用性。     

水体病毒浓缩条件的优化

病毒浓缩条件的优化是水环境病毒污染监测及灭活去除效率评价的基础,本文开发了在待浓缩水样中预先加入钠化蒙脱石吸附病毒以增加其沉降性能,并在优化絮凝条件下用聚合氯化铝絮凝沉淀蒙脱石钠的水体病毒浓缩方法.该方法对人为污染的脊髓灰质炎病毒(Poliovirus 1, PV1)、烟草花叶病毒(Tobacco mosaic virus, TMV)、大肠杆菌噬菌体(Phage of Ecoli, E.cp)在闽江水、生活污水、自来水中分别有高于84.3%的浓缩回收率,说明该浓缩方法具有较广泛的适用性和应用前景,可为水体病毒污染监测及灭活去除评价提供一个简便、回收率高、成本低的浓缩方法.     

环境水体中GⅡ型诺如病毒浓缩研究

自行设计一种适合野外现场采样的病毒采集浓缩仪,采用一种新型阳离子膜NanoCeram,进行环境水体中GⅡ型诺如病毒浓缩研究。通过预实验,考察了预处理膜、不同的二次浓缩方法、不同洗脱液对病毒回收率的影响,随后优化整个浓缩过程并确定最佳的病毒浓缩方法。结果显示,预处理膜对病毒回收影响较大,PEG-NaCl沉淀、硅藻土吸附这两种二次浓缩方法的效果相当,选择0.15mol/L Na2HPO4作为硅藻土洗脱液。确定了NanoCeram阳离子膜的病毒回收率为3.02%。最后对北京市丰台区洋桥生活污水进行现场采样,成功检测到GⅡ型诺如病毒,证明该方法有效可行,适合于环境水体中诺如病毒的浓缩分离和检测。     

水体病毒浓缩条件的优化

病毒浓缩条件的优化是水环境病毒污染监测及灭活去除效率评价的基础,本文开发了在待浓缩水样中预先加入钠化蒙脱石吸附病毒以增加其沉降性能,并在优化絮凝条件下用聚合氯化铝絮凝沉淀蒙脱石钠的水体病毒浓缩方法。该方法对人为污染的脊髓灰质炎病毒(Poliovirus 1,PV1)、烟草花叶病毒(Tobacco mosaic virus,TMV)、大肠杆菌噬菌体(Phage of Ecoli,E．cp)在闽汀水、生活污水、自来水中分别有高于84．3％的浓缩回收率,说明该浓缩方法具有较广泛的适用性和应用前景,可为水体病毒污染监测及灭活去除评价提供一个简便、回收率高、成本低的浓缩方法。     

基于磁珠吸附的污水病毒高通量富集方法研究

基于环境污水的传染病病原监测是基于病例的传染病监测体系的重要补充。当前常规的污水病毒富集方法存在人力耗费大、周期长、精密度低等缺陷，导致污水病原监测难以大规模开展。本研究系统地探究了磁珠吸附法（磁珠法）对污水中的传染病相关病毒（包括新冠状病毒（SARS-CoV-2）、诺如病毒（Norovirus, NoV））富集的回收率和精密度，探讨了不同样本存储条件对磁珠法富集病毒效果的影响，采取磁珠法和现行标准的聚乙二醇（PEG）沉淀法富集含不同SARS-CoV-2拷贝数加标污水中的病毒，并检测SARS-CoV-2的核酸浓度，通过计算病毒载量来比较两种方法的检出限、病毒回收率和重复富集的精密度；利用2023年5月-6月广东省五个地市采集的28份污水样本，比较两种方法对新鲜水样本中SARS-CoV-2、NoV的富集效果，并探索了污水经冻融以及4度保存24 h后，磁珠法富集SARS-CoV-2和NoV效果的差异。结果表明在加标水重复试验中，磁珠法的检出限在100拷贝/mL，而经PEG沉淀法富集的100拷贝/mL加标水中仅有一份检出阳性，磁珠法病毒回收率在1000拷贝/mL加标水中的变异系数是0.14，...     

水体病毒钙离子絮凝浓缩新方法研究

研究建立了一种从水体中浓缩病毒的新方法,即钙离子絮凝-柠檬酸缓冲液洗溶法,该方法的要点是先用一定量的钙离子溶液和钙离子絮凝剂絮凝水体中的病毒,再用pH5.0的0.3mol/L的柠檬酸缓冲液洗溶,然后再进一步超滤浓缩。此法可方便地将水体中的病毒浓缩10000倍以上。应用该法分别对人工播种于饮用水中的f2噬菌体和脊髓灰质炎疫苗病毒(PV1)进行了浓缩,结果发现f2噬菌体的平均回收率达96%,而PV1的回收率为100%,均显著高于阳电膜过滤法(P<0.05)。该方法快速、简便、有效。     

重组逆转录病毒浓缩方法研究

选择含有新霉素磷酸转移酶Ⅱ（Neor)的重组逆转录病毒载体,通过包装细胞进行包装,得到了含有重组逆转录病毒粒子的病毒溶液,该病毒溶液分别采用差速离心和滤膜截留两种方法进行浓缩,浓缩前,后的病毒溶液体外感染靶细胞NIH3T3以测定其滴度。结果显示,差速离心法的浓缩效率要优于滤膜截留法的浓缩效率,其浓缩效率能达到34．5％。     

水标本中脊髓灰质炎病毒三种浓缩方法的比较及改进方法的建立

本文用三种浓缩方法对人工污染标本进行了病毒回收率的比较试验,三种方法结果基本相同(47—49％)。在被检脊髓灰质炎Ⅰ型病毒蒸馏水标本中加1％NaCl可提高滤膜法对水中病毒的回收率;在含脊髓灰质炎病毒的河水、污水标本中加1％NaCl及AlCl_3(河水为0.0005mol/L、污水为0.001mol/L),离心沉淀过程中,我们惊奇地发现病毒全在沉渣中,结果表明用该方法病毒回收率竟提高了一倍(92—99％)。     

TMV在不同水体与温度条件下的灭活动力学

了解植物病毒在不同水体与温度条件下的灭活规律具有重要的理论与实际意义.本文以典型植物病毒烟草花叶病毒(TMV)为模型,比较了其在不同温度条件下,在闽江水、自来水、生活污水、微孔滤膜过滤除菌污水及超纯水中的灭活动力学.结果显示,温度是导致TMV灭活的重要因素,水温升高,病毒灭活速率加快;此外,某些水质因子也影响TMV的灭活效率,其中可溶性盐的存在及其含量对TMV的灭活会因所处的环境不同而异;某些微生物或代谢产物对植物病毒TMV具有灭活作用,而能生化降解的有机质加速TMV灭活可能是通过促进水体中的微生物增殖而起作用.     

Comparison of Concentration Methods for Quantitative Detection of Sewage-Associated Viral Markers in Environmental Waters

Pathogenic human viruses cause over half of gastroenteritis cases associated with recreational water use worldwide. They are relatively difficult to concentrate from environmental waters due to typically low concentrations and their small size. Although rapid enumeration of viruses by quantitative PCR (qPCR) has the potential to greatly improve water quality analysis and risk assessment, the upstream steps of capturing and recovering viruses from environmental water sources along with removing PCR inhibitors from extracted nucleic acids remain formidable barriers to routine use. Here, we compared the efficiency of virus recovery for three rapid methods of concentrating two microbial source tracking (MST) viral markers human adenoviruses (HAdVs) and polyomaviruses (HPyVs) from one liter tap water and river water samples on HA membranes (90 mm in diameter). Samples were spiked with raw sewage, and viral adsorption to membranes was promoted by acidification (method A) or addition of MgCl₂ (methods B and C). Viral nucleic acid was extracted directly from membranes (method A), or viruses were eluted with NaOH and concentrated by centrifugal ultrafiltration (methods B and C). No inhibition of qPCR was observed for samples processed by method A, but inhibition occurred in river samples processed by B and C. Recovery efficiencies of HAdVs and HPyVs were ∼10-fold greater for method A (31 to 78%) than for methods B and C (2.4 to 12%). Further analysis of membranes from method B revealed that the majority of viruses were not eluted from the membrane, resulting in poor recovery. The modification of the originally published method A to include a larger diameter membrane and a nucleic acid extraction kit that could accommodate the membrane resulted in a rapid virus concentration method with good recovery and lack of inhibitory compounds. The frequently used strategy of viral absorption with added cations (Mg²⁺) and elution with acid were inefficient and more prone to inhibition, and will result in underestimation of the prevalence and concentrations of HAdVs and HPyVs markers in environmental waters.     

Detection of viruses in drinking water by concentration on magnetic iron oxide.

Discharge of raw domestic wastes containing human enteric viruses into water courses, consumption of untreated water from canals, streams, and shallow wells in villages, and cross-contamination of water in the distribution system because of intermittent water supply in urban areas continue to cause widespread outbreaks of infectious hepatitis in India. To detect a low number of viruses in 50- to 100-liter samples of water, a method was developed with magnetic iron oxide as the virus adsorbent. Poliovirus-seeded dechlorinated tap water, adjusted to pH 3.0 and 0.0005 M AlCl3, was filtered through a 10-g bed of iron oxide sandwiched between two AP20 prefilter pads held in a 142-mm-diameter, stainless-steel holder. Virus was eluted from iron oxide by recirculating three times a 100-ml volume of 3% beef extract, pH 9.0. The eluate was reconcentrated to 5 ml by adjusting to pH 3, adding 1 g of iron oxide, stirring for 30 min, and eluting the readsorbed virus with 5 ml of beef extract, pH 9.0. Virus recovery varied from 60 to 80%. Using the above method, we took a survey of drinking water at three locations in Nagpur during 1976 and found the presence of virus in 7 of 50 samples. The quantity of virus recovered ranged from 1 to 7 plaque-forming units per 30 to 60 liters. Virus was detected in some samples even with residual chlorine. No coliforms were detected in the virus-positive samples.     

Recovery of Small Quantities of Viruses from Clean Waters on Cellulose Nitrate Membrane Filters

A method is described for quantitatively recovering small amounts of viruses from large volumes of buffered, distilled water. Development of the method was motivated by the anticipated need for testing large volumes of renovated sewage for viruses. The method consists of adsorbing viruses onto cellulose nitrate membrane filters (0.45 mum pore size) from water containing sufficient Na(2)HPO(4) to produce a molarity of 0.05 and sufficient citric acid to produce a pH of 7, and eluting the adsorbed viruses in 3% beef extract under extended sonic treatment. Complete recovery of poliovirus 1, echovirus 7, and coxsackievirus B3 resulted when less than 100 plaque-forming units were added to 1-liter quantities of water. Recoveries of reovirus 1 were almost as good. Preliminary studies indicate that good recoveries can be made from 25-gal quantities of water. The method described is efficient in waters of high quality and may be useful for recovering viruses in renovated, and perhaps in tap waters, but not in waters containing certain organic matter unless that matter is first removed.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters.

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous concentration of four enteroviruses from tap, waste, and natural waters

The efficiency of virus recovery from water was investigated by using a method which enabled the concentration of a mixture of four enteroviruses with determination of their individual recovery efficiencies. The four viruses used (poliovirus 1, coxsackievirus A9, coxsackievirus B1, and echovirus 7) represented each of the four major subgroups of enteroviruses. This method, which was based on selective antibody neutralization, was used to investigate the effects of input water quality on enterovirus concentration by Balston filters (grade C; Balston, Inc., Lexington, Mass.) and organic flocculation. With tap water, the average recovery efficiency of the four viruses was 97%. Concentration from natural waters, including samples from two lakes (Lake Kinneret and the Hula Nature Reserve) and the Mediterranean Sea, resulted in similarly high average recovery efficiencies. Echovirus 7 was recovered with a slightly lower average efficiency from these types of water than were the other viruses. In comparison with other types of water, virus concentration from Jerusalem wastewater generally had a slightly lower efficiency of recovery, ranging from 63 to 75% for each of the viruses, with an overall average of 68%. The ability of each concentration step, membrane filtration or organic flocculation, to recover the viruses from water was assayed. For the filtration step, although there were not large differences in virus recoveries from tap water, echovirus 7 was recovered with the lowest efficiency (72%), and poliovirus 1 was recovered with the highest (87%) efficiency. Overall virus recovery by the filtration step was least efficient for wastewater (73%) and most efficient for seawater (107%).(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.     

Evaluation of various soil water samplers for virological sampling.

Two commercially available soil water samplers and a ceramic sampler constructed in our laboratories were evaluated for their ability to recover viruses from both tap water and secondary sewage effluent. The ceramic sampler consistently gave the best recoveries of viruses from water samples. Soil columns containing ceramic samplers at various depths provide a simple method for studying virus transport through sewage-contaminated soils.     

Evaluation of various soil water samplers for virological sampling

Two commercially available soil water samplers and a ceramic sampler constructed in our laboratories were evaluated for their ability to recover viruses from both tap water and secondary sewage effluent. The ceramic sampler consistently gave the best recoveries of viruses from water samples. Soil columns containing ceramic samplers at various depths provide a simple method for studying virus transport through sewage-contaminated soils.