两种水源性诺如病毒富集方法的比较

评估阳离子膜吸附-洗脱法和阴离子膜吸附-洗脱法在富集水源性诺如病毒（NV）过程中的病毒回收效果、消耗时间及使用成本,可以获得更适用于水源性诺如病毒实际监测工作的较优富集方法.以无锡市疾病预防控制中心保存的诺如病毒阳性标准质粒为模板,采用荧光定量RT-PCR（反转录酶-聚合酶链反应）方法绘制病毒定量标准曲线,并应用于病毒的绝对定量.将含诺如病毒的粪便悬液先稀释成3个不同浓度,并加入灭菌水中,分别经过阴离子膜（硝酸纤维素滤膜）或阳离子膜（Nanoceram膜）吸附洗脱,最终相同体积洗脱液提取核酸后用荧光定量RT-PCR方法进行绝对定量,并进一步计算最终病毒回收率.结果表明:①样本病毒浓度为8×101~8×1010 copies/μL时,病毒拷贝数的lg对数值与C_t（荧光定量PCR中达到设定阈值所经过的循环数）呈线性关系.②随着样本病毒浓度的增加,阴离子膜法和阳离子膜法的病毒回收率都相应增加.③当检测样本中病毒浓度分别为8×101、4×102 copies/μL时,阴离子膜法回收率分别为22.2%±4.1%和36.8%±6.2%,阳离子膜法回收率分别为17.4%±1.5%和28.8%±6.1%,说明阴离子膜吸附-洗脱法对病毒的富集回收率显著高于阳离子膜吸附-洗脱法（P < 0.05）.研究显示,综合富集效果、耗费时间和成本等因素,阴离子膜吸附-洗脱法比阳离子膜吸附-洗脱法更适用于日常水源性诺如病毒的富集工作.      

污水中肠道病毒及肠道致病菌的同步快速检出法

取池塘水5000毫升,pH6.8,经棉花过滤后再经直径150毫米,孔径3微米的微孔滤膜(上海医药工业研究院制备)减压过滤,煮沸灭菌,以便除掉颗粒物及水样中偶然可能存在的肠道病毒及肠道致病菌。凉后加试验病毒(小儿麻痹病毒1型无毒株,昆明医学生物学研究所)1毫升(TCID_(50)为6.50)。再添加经培养过液的甲型副伤寒菌5000个,用无菌玻璃棒搅匀。以上为模拟的含有肠道微生物的污水水样。一、水样中病毒的浓缩用12N的HCI调pH至3.5后,立即减压过滤。使通过直径150毫米,孔径0.45微米的过滤膜,使病毒吸附于膜面。洗脱病毒将过滤器自抽气瓶解下,移换另一支灭菌抽气瓶使之密闭,打开真空泵,自漏斗加     

饮用水源水中肠道病毒的检测方法

介绍饮用水源水中肠道病毒的检测方法。采用滑石粉 -硅藻土吸附浓缩水样中的肠道病毒 ,以 3 %牛肉浸膏洗脱 ,将病毒接种于敏感细胞中 ,根据细胞在病毒作用下的病变效应即空斑形成单位 ( PF L) ,定量计算水中病毒含量。     

利用毛细管气相色谱法测定水和废水中的丙烯酰胺

本文采用毛细管色谱法建立了水和废水中丙烯酰胺的测定程序。以活性炭柱吸附、浓缩样品中的丙烯酰胺，用甲醇洗脱，洗脱液用FID测定。方法回收率为83．1～99．0％，相对标准偏差为5．34％，检出限为0．016mg／L。     

水中悬浮物测定方法探讨

根据国家测试废水中悬浮物浓度的分析标准,在采用不同生产厂家生产的微孔滤膜（孔径0.45？m,直径50mm）测试煤矿矿井水时,常碰到比较清洁的水样容易出现负值的现象。通过对微孔滤膜的反复实验并深入分析,提出了如何改进＂滤膜准备＂的操作步骤建议,对更加真实反映水体中悬浮物浓度具有一定意义。     

D301弱碱性阴离子交换树脂吸附Cr(Ⅵ)的研究

实验探索性地采用D301弱碱性阴离子交换树脂去除重金属废水中的C(rⅥ),为开发出一种高效、安全的处理、富集和回收含C(rⅥ)废水的新工艺提供理论和实验依据。静态吸附实验结果表明,该树脂对C(rⅥ)的吸附量随pH的降低而增加,在pH=2时吸附量为17.54 mg/g;吸附平衡过程符合Freundlich方程,且最大吸附量为94.34 mg/g;动力学和热力学研究表明,吸附过程为颗粒扩散控制,自发且放热的,表观吸附活化能为E(a300 mg/L)=16 243.06 kJ/mol;树脂的再生能力强,经过3次再生,树脂的平衡吸附量仅下降了8.54%;动态吸附和洗脱实验表明,以5%NaOH溶液为洗脱剂效果最佳,洗脱液浓度高达6 918.74 mg/L,浓缩了约69倍,且动态洗脱曲线没有托尾现象;该树脂尤其适合处理低浓度含C(rⅥ)废水(≤100 mg/L),且几乎不受流速限制。     

膜生物反应器对白斑综合症病毒的去除研究

研究了不同孔径的膜生物反应器对游离白斑综合症病毒(white spot syndrome virus,WSSV)的截留效果,对不同膜生物反应器的进水、上清液、出水进行荧光定量PCR检测.结果表明,0.45 μm孔径的膜组件对磷酸缓冲液中WSSV的去除量仅0.6 lg;0.22 μm孔径的膜对WSSV病毒的去除量为1.18 lg;0.1 μm孔径的膜对WSSV的去除量为5.5 lg.进一步研究表明,在养殖废水中添加游离的WSSV时,随着时间的延长,膜组件的生物样物质对WSSV去除起了重要的作用;孔径为0.45 μm的膜生物反应器,对WSSV的去除量也达到5.35 lg;3种不同孔径膜生物反应器中运行12h后,对WSSV的去除率并没有显著差异.     

污水再生处理系统中隐孢子虫和贾第鞭毛虫检测方法的优化

隐孢子虫和贾第鞭毛虫是2种严重危害水质安全的病原性原生动物.与USEPA1623方法相比,以膜过滤-洗脱法作为浓缩方式的检测流程回收率较高且成本较低.本研究通过对膜过滤-洗脱环节和免疫磁性分离(IMS)过程进行改进和参数优化,建立了适用于污水再生处理系统的两虫检测方法.研究发现,对滤膜进行刮擦处理后隔夜浸泡,以及洗脱前剧烈振荡等操作能够显著提高并稳定回收率.投加高岭土浊液提高水样的浊度至4NTU更加有效地提高了低浊水样的浓缩回收率.离心浓缩后洗涤沉淀并进行2次酸解离是降低IMS过程水质干扰的有效措施.采用该优化方法对不同来源水样进行检测,隐孢子虫回收率超过70%,贾第鞭毛虫回收率超过80%,明显高于1623方法的接受标准(>24%).     

阴离子型聚丙烯酰胺在离子交换膜上的吸附规律

研究了阴离子交换膜对阴离子型聚丙烯酰胺的吸附规律和影响因素.采用静态吸附的方法,测定了不同温度、不同浓度的阴离子型部分水解聚丙烯酰胺(HPAM)溶液在阴离子交换膜上的吸附量、吸附平衡时间,考察了初始HPAM溶液浓度、pH值以及溶液中其他离子浓度对其在离子交换膜上吸附的影响,目的是阐明阴离子交换膜对HPAM分子的吸附动力学过程、探讨各影响因素对吸附过程的影响.结果表明,阴离子型部分水解聚丙烯酰胺在阴离子交换膜上有明显的吸附作用,但在阳离子交换膜上吸附量几乎为0;阴离子交换膜对聚合物的吸附平衡时间随聚合物溶液初始浓度的增大而延长,且不同浓度、不同温度下的吸附过程动力学特征都能很好地遵循准二级动力学模型;303、308和313 K温度下,阴离子交换膜对聚合物的等温吸附可用Freundlich等温吸附模型很好地拟合,相关系数R2均达到0.99以上,温度越高,吸附量越大;聚合物溶液pH值和离子浓度对吸附效果有显著影响:pH=6时,吸附量达到最大值;吸附量随着离子浓度的增加而增大.     

富集甲基汞的树脂膜筛选及条件优化

树脂膜作为DGT技术的核心部分,是影响 DGT测定的关键因素。为确保树脂吸附和洗脱实验的准确性,筛选出醋酸-盐酸混合溶液作为甲基汞溶液的保存试剂。以标液中甲基汞的吸附率为指标,比较了4种吸附树脂的基本特征及在室内标液浓度为1μg/L的条件下对甲基汞的吸附效果,筛选出吸附效果最佳的树脂TCH-95,并对TCH-95树脂胶的不同洗脱试剂进行了研究。结果表明：24h TCH-95对甲基汞的吸附率高达98.9%。且在洗脱实验中,对硫脲-盐酸混合溶液添加冰醋酸有助于提高树脂胶中甲基汞的洗脱率,其2%硫脲-0.2mol/L HCl-4%HAc混合溶液的甲基汞洗脱率为84.5%,是目前TCH-95树脂的最佳洗脱剂。     

Concentration and Recovery of Viruses from Water: A Comprehensive Review

Enteric viruses are a cause of waterborne disease worldwide, and low numbers in drinking water can present a significant risk of infection. Because the numbers are often quite low, large volumes (100–1,000 L) of water are usually processed. The VIRADEL method using microporous filters is most commonly used today for this purpose. Negatively charged filters require the addition of multivalent salts and acidification of the water sample to effect virus adsorption, which can make large-volume sampling difficult. Positively charged filters require no preconditioning of samples, and are able to concentrate viruses from water over a greater pH range than electronegative filters. The most widely used electropositive filter is the Virosorb 1MDS; however, the Environmental Protection Agency has added the positively charged NanoCeram filters to their proposed Method 1615. Ultrafilters concentrate viruses based on size exclusion rather than electrokinetics, but are impractical for field sampling or processing of turbid water. Elution (recovery) of viruses from filters following concentration is performed with organic (e.g., beef extract) or inorganic solutions (e.g., sodium polyphosphates). Eluates are then reconcentrated to decrease the sample volume to enhance detection methods (e.g., cell culture infectivity assays and molecular detection techniques). While the majority of available filters have demonstrated high virus retention efficiencies, the methods to elute and reconcentrate viruses have met with varying degrees of success due to the biological variability of viruses present in water.     

Effectiveness of Poliovirus Concentration and Recovery from Treated Wastewater by Two Electropositive Filter Methods

Enteric viruses are often present in low numbers in various water matrices. Virus sampling therefore involves multiple concentration steps to condense large samples down to small volumes for detection by cell culture or molecular assays. The NanoCeram^® Virus Sampler has been demonstrated to be effective for the recovery of viruses from tap water, surface waters, and seawater. The goal of this study was to evaluate a new method using NanoCeram^® filters for the recovery of poliovirus 1 (PV-1) from treated wastewater. Activated sludge effluent samples were spiked with PV-1 and concentrated in side-by-side tests by two methods: (1) NanoCeram^® filtration, elution with sodium polyphosphate buffer, secondary concentration via centrifugal ultrafiltration; and (2) 1MDS filtration, elution with beef extract, secondary concentration via organic flocculation. The virus retention and elution efficiencies did not differ significantly between the two methods. In contrast, the secondary concentrate volume was smaller for the NanoCeram^® method (8.4 vs. 30 mL) and the secondary concentration efficiencies were different between the two methods with 98 % for centrifugal ultrafiltration (NanoCeram^®) and 45 % for organic flocculation (1MDS). The overall method efficiencies were significantly different (P ≤ 0.05) with the NanoCeram^® method yielding a 57 % and the 1MDS a 23 % virus recovery. In addition, there appeared to be less interference with viral detection via polymerase chain reaction with the NanoCeram^® concentrates. This NanoCeram^® method therefore is able to efficiently recover PV-1 from large volumes of wastewater and may serve as an inexpensive alternative to the standard 1MDS filter method for such applications.     

逆转录-聚合酶链反应(RT-PCR)技术同时检测水中多种肠道病毒

建立了一种利用通用引物RT-PCR技术检测水中肠道病毒的方法.利用脊髓灰质炎病毒1～3型,柯萨奇病毒B3型作为参考病毒株,根据肠道病毒RNA5′非编码区中具有高度同源性的序列来设计通用引物.比较了M-MLV酶和AMV酶的逆转录效果,AMV酶能够成功地从地表水和生活污水中逆转录病毒RNA,更适于实际应用.对比研究了PCR过程中的退火温度,c(Mg2＋)等因素对RT-PCR检测结果的影响,选择退火温度55　℃,c(Mg2＋)为2　mmol/L的反应条件,优化了RT-PCR检测方法.通过检测水样中接种的连续稀释的病毒,确定了该检测方法的灵敏度为38　CCID₅₀.考察人工污染的地表水、污水、二级处理出水样品发现,检测灵敏度基本一致.该方法可应用在实际环境的肠道病毒检测中.      

Use of Preservative Agents and Antibiotics for Increased Poliovirus Survival on Positively Charged Filters

Environmental surveillance of poliovirus (PV) and other non-enveloped viruses can help identify silent circulation and is necessary to certify eradication. The bag-mediated filtration system is an efficient method to filter large volumes of environmental waters at field sites for monitoring the presence of viruses. As filters may require long transit times to off-site laboratories for processing, viral inactivation or overgrowth of bacteria and fungi can interfere with virus detection and quantification (Miki and Jacquet in Aquatic Microb Ecol 51(2):195–208, 2008). To evaluate virus survival over time on ViroCap^? filters, the filters were seeded with PV type 1 (PV1) and/or MS2 and then dosed with preservatives or antibiotics prior to storage and elution. These filters were stored at various temperatures and time periods, and then eluted for PV1 and MS2 recovery quantification. Filters dosed with the preservative combination of 2% sodium benzoate and 0.2% calcium propionate had increased virus survival over time when stored at 25 °C, compared to samples stored at 25 °C with no preservatives. While elution within 24 h of filtration is recommended, if storage or shipping is required then this preservative mixture can help preserve sample integrity. Addition of an antibiotic cocktail containing cephapirin, gentamicin, and Proclin^? 300 increased recovery after storage at 4 and 25 °C, when compared to storage with no antibiotics. The antibiotic cocktail can aid sample preservation if access to appropriate antibiotics storage is available and sample cold chain is unreliable. This study demonstrated that the use of preservatives or antibiotics is a simple, cost-effective method to improve virus detection from ViroCap cartridge filters over time.     

Simultaneous Concentration of Enteric Viruses and Protozoan Parasites: A Protocol Based on Tangential Flow Filtration and Adapted to Large Volumes of Surface and Drinking Waters

A filtration system, based on tangential flow filtration (TFF) followed by ultracentrifugation was developed in order to concentrate simultaneously viruses and parasites from large volumes of water. For TFF, no pre-treatment of the membrane is performed but a post-rinsing step using high pH-beef extract-based eluant. Applying our protocol to 20 l of surface waters spiked with vaccinal poliovirus-1, ϕX174 and MS2 bacteriophages resulted in an averaged viral recovery of 75% by TFF and 91% by ultracentrifugation (total viral recovery of 70%). Our protocol was further applied to 31 environmental samples including surface, ground and drinking waters from the Grand Duchy of Luxembourg in order to assess the occurrence of protozoan parasites (Cryptosporidium parvum and Giardia lamblia (oo)cysts), pathogenic viruses (enterovirus, norovirus and adenovirus) and infectious bacteriophages (somatic coliphages and F-specific phages) in these samples. High viral recovery rates of > 70% were confirmed concentrating environmental strains of somatic and F-specific coliphages from non-spiked surface waters. Parasites and enteric viruses were detected in 86 and 40% of the surface waters used for drinking water production, respectively. Infectious bacteriophages were isolated from all surface waters and in two out of seven (29%) groundwaters revealing a susceptibility of the corresponding wells to viral pollution. TFF-based method proved to be efficient for surveying the occurrence of non-bacterial pathogens such as enteric viruses and protozoan parasites in large volumes of environmental waters.     

Improvement of detection method of Cryptosporidium and Giardia in reclaimed water

Cryptosporidium and Giardia are two typical species of pathogenic protozoans that seriously endanger water quality. Previous works indicated that detection of Cryptosporidium and Giardia with modified United States Environmental Protection Agency (USEPA) method-1623 using a membrane filtration-elution for sample concentration attained better recovery and lower cost compared to the USEPA method-1623. Several improvements of membrane filtration-elution step as well as immunomagnetic separation (IMS) steps were investigated and an optimized method for detection of Cryptosporidium and Giardia in wastewater reclamation system was recommended in this paper. The experimental results show that an overnight soak of the membrane after scraping and vortex agitation before elution could enhance and stabilize the recovery. Increasing turbidity to 4 NTU by adding kaolin clay before filtration could effectively improve the recovery of low-turbidity water. Washing the concentrate after centrifugation and twice acid dissociation both reduced the impact of water quality to protozoan recovery. Protozoans in different water samples were determined by this optimized method, and the recovery of Cryptosporidium and Giardia were above 70% and 80% respectively, much higher than the acceptance of method-1623. __________ Translated from Environmental Science, 2006, 27(12): 2547–2552 [译自: 环境科学]     

Application of Acidic Elution to Virus Concentration Using Electropositive Filters

The effect of the type and pH of an elution solution on the recovery of poliovirus from water by a virus concentration method using an electropositive filter was evaluated. The experimental results obtained indicated the potential usefulness of H₂SO₄ (pH 1.5–3.5) as a novel solution for virus elution.     

Detection of Enteroviruses in Influent and Effluent Flow Samples from Wastewater Treatment Plants in Italy

This study evaluated the presence and seasonal distribution of polio and other enteroviruses in four wastewater treatment plants in three cities in Italy, using different treatment systems. Detection of enteroviruses was carried out by virus isolation in cell cultures after concentration of water samples collected at both inlet and outlet of the treatment plants, following the methods described in the WHO guidelines. Viral serotypes isolated before and after water treatment were compared. Forty-eight non-polio enteroviruses were isolated from 312 samples collected at the inlet of the four wastewater treatment plants, 35 of which were Coxsackievirus type B (72.9 %) and 13 Echovirus (27.1 %). After treatment, 2 CVB3, 1 CVB5, and 1 Echo 6 were isolated. CVB3 and Echo 6 serotypes were also detected in samples collected at the inlet of the TP, in the same month and year. The high rate of detection of infectious enteroviruses in inlet sewage samples (30.1 %) indicates wide diffusion of these viruses in the populations linked to the collectors. The incomplete removal of infectious viruses following sewage treatment highlights possible risks for public health relate to treated waters discharge into the environment.