饮用水中隐孢子虫qPCR检测研究

分子生物学的发展推动了分子技术在病原体检测诊断中的应用,建立成熟可靠的饮用水中隐孢子虫的分子检测方法也迫在眉睫.本研究直接提取饮用水浓缩液DNA,对隐孢子虫卵囊目的基因进行定量PCR(quantitative PCR,简称qPCR)检测,并对该体系进行了综合优化.本研究比较了不同冻融处理及不同DNA提取试剂盒对隐孢子虫卵囊DNA提取效果,结果显示,QIAamp DNA Mini kit配合冻融前处理(液氮/沸水5个循环)提取DNA含量较高,用这种方法可以提取到(4±1)个隐孢子虫卵囊DNA.本研究比较了隐孢子虫SYBR green染料法及Taqman探针法qPCR,结果显示,探针法和染料法均可以检测到10个18S rRNA基因拷贝(一个隐虫卵囊含有20个18S rRNA基因拷贝).采用该检测体系测定的去离子水和水源水中隐孢子虫的加标回收率分别达到了55%和46%;本研究提供了饮用水中隐孢子虫检测的一种可行性分子方法.     

水中的隐孢子虫卵囊及其检测与灭活

介绍了隐孢子虫卵囊（ＣＳＯ）的介水传播情况，对目前国内外检测，灭活水隐孢子虫卵囊的最新研究进展了比较全面的介绍。     

隐孢子虫病与饮用供水

本文简介了人们对隐孢子虫、隐孢子虫病以及隐孢子卵囊污染环境方面的基本认识,对隐孢子卵囊污染的控制和给水处理工艺改进方面作了重点论述。     

控制水体隐孢子虫的研究进展

介绍了隐孢子虫病的症状和传播途径 ,并着重论述控制水体隐孢子虫卵囊的化学灭活方法。     

紫外/超声协同灭活隐孢子虫的影响因素及机制

为研究UV/US（Ultraviolet/Ultrasonic,紫外/超声）协同对水中隐孢子虫的灭活机制,采用UV灯（功率为14 W）与US发生器（频率为20 kHz,功率为150 W）组合装置协同灭活隐孢子虫,考察pH、温度、浊度和HA（腐殖酸）对UV/US协同灭活隐孢子虫的影响,并通过SEM（扫描电镜）、蛋白质试验和琼脂糖凝胶电泳检测对灭活机制进行了探讨.结果表明:pH对UV/US杀灭隐孢子虫的影响不大,碱性条件下灭活率略高于中性和酸性条件;温度对灭活率有一定影响,5℃下灭活率较低,随温度的上升,灭活率逐渐提高,25℃下10 min灭活率可达99%以上;悬浮物抑制隐孢子虫的灭活,浊度为40 NTU时,UV/US作用25 min的灭活率仅为93.88%;HA对灭活的影响表现为低浓度促进,高浓度抑制;ρ（HA）高于10 mg/L时,继续增大ρ（HA）对隐孢子虫灭活率影响不大.研究显示:UV/US协同作用对隐孢子虫的灭活机制主要是使其卵囊破裂,同时损伤了隐孢子虫胞内的DNA.      

超声灭活水中隐孢子虫影响因素及机制

采用超声灭活隐孢子虫卵囊,超声频率为19.8kHz,功率为151W,温度控制在(20±1)℃, 探讨了浊度、ρ(腐殖酸)及无机离子质量浓度对超声灭活隐孢子虫的影响,并通过扫描电镜观察及动力学分析探究了超声灭活隐孢子虫的机制. 结果表明:在浊度为0~21.46NTU范围内,隐孢子虫的灭活率先升后降,浊度为1.13NTU时灭活率最高,为94.7%;隐孢子虫灭活率随ρ(腐殖酸)的增加而降低,当ρ(腐殖酸)为50.0mg/L时灭活率降至78.4%. Cu2+、Mn2+和SO₄2-对灭活率影响较小,当ρ(Cu2+)为0.5mg/L时,隐孢子虫灭活率(93.2%)比未添加Cu2+时增加了0.7%;ρ(CO₃2-)增加,隐孢子虫灭活率下降,当ρ(CO₃2-)为150mg/L时灭活率降至82.5%. 扫描电镜形态学观察表明,超声可破坏隐孢子虫细胞结构;动力学分析表明,自由基氧化作用对灭活率的贡献为15.6%,说明超声灭活隐孢子虫的主要机制为空化作用产生的机械剪切作用.      

隐孢子虫和贾第鞭毛虫的危害及其控制技术

病原微生物对水体的污染以及在饮用水中的存在是一个重要的公共卫生问题.微小隐孢子虫和蓝氏贾第鞭毛虫(简称两虫)被认为是世界上最主要的导致人体腹泻的水源性原生动物寄生虫,隐孢子虫卵囊和贾第鞭毛虫孢囊具有个体微小,致病量低、抵抗环境选择性压力强、易于造成两虫病暴发流行等特点,对公共健康及公共安全具有潜在的风险.饮用水处理中,...     

隐孢子虫病及其水媒传播控制

综述了隐孢子虫和隐孢子虫病的特性，症状和传播途径，水媒传播的重要案列及其为水工业和水源保护带来的启示。介绍了隐孢子虫的检测方法和相应水质指标的研究现状；给水工业中控制隐孢子虫病传播的主要手段。     

二氧化氯灭活水中隐孢子虫的影响因素及机理研究

以荧光活体染色法研究了ClO2浓度、灭活时间、浊度、pH值、温度、有机物含量等,对ClO2灭活隐孢子虫效果的影响,并利用扫描电镜和蛋白质实验初步探究了灭活机理.结果显示,当pH7.0,水温为25℃,浊度为1NTU时,投加3mg/L ClO2经过120min,可以达到最适消毒效果(存活率小于1%),隐孢子虫的灭活率与ClO2投加浓度、作用时间成非线性正相关.浊度是影响ClO2灭活隐孢子虫的主要因素,浊度越低,灭活效果越佳;水温(较)低,灭活效果稍差;酸性较于碱性更适宜ClO2灭活隐孢子虫;可溶性有机物一定程度上影响ClO2的灭活效果.扫描电镜和蛋白试验表明,ClO2主要破坏其细胞表面结构,从而引起隐孢子虫死亡.     

再生水利用的病原微生物浓度限值探讨

采用微生物定量风险评价的方法,结合北京市某城市污水回用工程,对再生水用于园林绿化、道路冲洗与降尘等市政用途时的病原微生物浓度限值进行了探讨.提出的限值为:大肠埃希氏杆菌70个/L、沙门氏菌0.5 CFU/L、志贺氏菌0.1CFU/100L、甲肝病毒0.001 PFU/100L、轮状病毒1.2×10^-3PFU/100L、脊髓灰质炎病毒0.07 PFU/100L、柯萨奇病毒0.04PFU/100L、埃可病毒0.05 PFU/100L、隐孢子虫卵囊0.1个/100L、贾弟鞭毛虫卵囊0.03个/100L.     

Evaluation of the infectivity, gene and antigenicity persistence of rotaviruses by free chlorine disinfection

The effects of free chlorine disinfection of tap water and wastewater effluents on the infectivity, gene integrity and surface antigens of rotaviruses were evaluated by a bench-scale chlorine disinfection experiments. Plaque assays, integrated cell culture-quantitative RT-PCR (ICC-RT-qPCR), RT-qPCR, and enzyme-linked immunosorbent assays (ELISA), respectively, were used to assess the influence of the disinfectant on virus infectivity as well as genetic and antigenic integrity of simian rotavirus SA11 as a surrogate for human rotaviruses. The ICC-RT-qPCR was able to detect rotaviruses survival from chlorine disinfection at chlorine dose up to 20 mg/L (60 min contact), which suggested a required chlorine dose of 5 folds (from 1 to 5 mg/L) higher than that indicated by the plaque assay to achieve 1.8 log₁₀ reductions in tap water with 60 min exposing. The VP7 gene was more resistant than the infectivity and existed at chlorine dose up to 20 mg/L (60 min contact), while the antigencity was undetectable with chlorine dose more than 5 mg/L (60 min contact). The water quality also impacted the inactivation efficiencies, and rotaviruses have a relatively higher resistant in secondary effluents than in the tap water under the same chlorine disinfection treatments. This study indicated that rotaviruses have a higher infectivity, gene and antigencity resistance to chlorine than that previously indicated by plaque assay only, which seemed to underestimate the resistance of rotaviruses to chlorine and the risk of rotaviruses in environments. Present results also suggested that re-evaluation of resistance of other waterborne viruses after disinfections by more sensitive infectivity detection method (such as ICC-RT-qPCR) may be necessary, to determine the adequate disinfectant doses required for the inactivation of waterborne viruses.     

淡水噬藻体及其他溶藻因子的分布与感染力

研究了不同时空条件下噬藻体的分布与效力的变化.2001年8～11月共采集了18个水样,对14种藻进行直接感染,从其中的5个水样中分离到了噬藻体(蓝藻病毒),它们主要来源于降温以前富营养化池塘的水样,同时分离得到了4株溶藻细菌.在接受直接感染的14种藻中,聚球藻、组囊藻、微囊藻、织线藻、鱼腥藻7120、衣藻及小球藻为敏感种类,而螺旋藻、念珠藻、鞘丝藻、颤藻、鱼腥藻595、鱼腥藻1444、栅藻则为不敏感种类,敏感种类中织线藻受到噬藻体的感染,聚球藻、微囊藻和织线藻受到溶藻细菌的溶解.从富营养化的池塘采集的水样溶藻能力最强,其次为清水(自来水源)池塘,而大中型湖泊与河流水样的溶藻效果最差.随着温度的降低,水样的感染能力下降.     

Effect of the Shellfish Proteinase K Digestion Method on Norovirus Capsid Integrity

Norovirus outbreaks are associated with the consumption of contaminated shellfish, and so efficient methods to recover and detect infectious norovirus in shellfish are important. The Proteinase K digestion method used to recover norovirus from shellfish, as described in the ISO 15216, would be a good candidate but its impact on the virus capsid integrity and thus infectivity was never examined. The aim of this study was to assess the impact of the Proteinase K digestion method, and of the heat treatment component of the method alone, on norovirus (genogroups I and II) and MS2 bacteriophage capsid integrity. A slightly modified version of the ISO method was used. RT-qPCR was used for virus detection following digestion of accessible viral RNA using RNases. MS2 phage infectivity was measured using a plaque assay. The effect of shellfish digestive glands (DG) on recovery was evaluated. In the presence of shellfish DG, a reduction in MS2 phage infectivity of about 1 log₁₀ was observed after the Proteinase K digestion method and after heat treatment component alone. For norovirus GII and MS2 phage, there was no significant loss of genome following the Proteinase K digestion method but there was a significant 0.24 log₁₀ loss of norovirus GI. In the absence of shellfish DG, the reduction in MS2 phage infectivity was about 2 log₁₀, with the addition of RNases resulting in a significant loss of genome for all tested viruses following complete Proteinase K digestion method and the heat treatment alone. While some protective effect from the shellfish DG on viruses was observed, the impact on capsid integrity and infectivity suggests that this method, while suitable for norovirus genome detection, may not completely preserve virus infectivity.     

Solar Disinfection of Water for Inactivation of Enteric Viruses and its Enhancement by Riboflavin

Solar disinfection (SODIS) has been described as a cheap and effective method of treating contaminated water to inactivate pathogenic microorganisms. In this study, SODIS was assessed for its efficacy in inactivating three enteric viruses (coxsackievirus B3, coxsackievirus B5 and poliovirus), either on its own or in the presence of riboflavin as a disinfection enhancer. On its own, SODIS produced a reduction of virus infectivity of 4–6 log₁₀ in 6 h. In the presence of riboflavin, inactivation was more rapid in all viruses studied, and with coxsackievirus B5 and poliovirus an extra 1–2 log₁₀ increase in reduction of infectivity was observed after 6 h exposure. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.     

Virus Genome Quantification Does not Predict Norovirus Infectivity After Application of Food Inactivation Processing Technologies

When determining the effect of food processing on the infectivity of any contaminating virus, it is necessary to distinguish unambiguously between infectious and non-infectious viruses present. However, this can be difficult in the particular case of noroviruses (NoVs) because no reliable cell culture model is available. The aim of this study was to assess the use of molecular methods—RT real-time PCR (RT-qPCR) and enzymatic treatment (ET) coupled to RT-qPCR—to quantify the infectivity of NoV after application of various inactivating food-processing technologies. RT-qPCR and ET-RT-qPCR gave significantly different (P < 0.01) results concerning the reduction in viral genome counts by all inactivation procedures and conditions used, except for HHP treatment at 600 MPa for 5 min. These findings indicate that the ET prior to RT-qPCR has an effect on the estimation of the reduction of virus genome counts, and may eliminate genomes of affected virus particles. However, no correlation was found between the results obtained by ET-RT-qPCR and those obtained by cell culture. Therefore, the effect is presumably only partial, and not adequate to allow accurate estimation of virus inactivation. Consequently, our results indicate that the quantification of virus genomes by PCR, regardless of prior ET, is not adequate for establishing virus inactivation and/or infectivity. In addition, our results also illustrate that the general effect of virus inactivation is not directly correlated to effects on the integrity of virus genome and protein capsid. Presumably, inactivation by food processing is the consequence of effects on proteins involved in adhesion and invasion stages.     

Capsid and Infectivity in Virus Detection

The spectacular achievements and elegance of viral RNA analyses have somewhat obscured the importance of the capsid in transmission of viruses via food and water. The capsid’s essential roles are protection of the RNA when the virion is outside the host cell and initiation of infection when the virion contacts a receptor on an appropriate host cell. Capsids of environmentally transmitted viruses are phenomenally durable. Fortuitous properties of the capsid include antigenicity, isoelectric point(s), sometimes hemagglutination, and perhaps others. These can potentially be used to characterize capsid changes that cause or accompany loss of viral infectivity and may be valuable in distinguishing native from inactivated virus when molecular detection methods are used.     

国外感染性废弃物的管理

感染性废气物对人们的身心健康有极大的威胁．然而从目前的实际情况来看，相当一部分是与一般城市生活垃圾混合在一起处理的情况较常见。鉴于上述情况．文中将欧美日发达国家对感染性废气物的收集．管理及处理情况给予介绍。     

Survival of Human Adenovirus 41 in Land-Applied Manure and Biosolids

Human Adenovirus 41 (Ad41) is an important human enteric pathogen and widely prevalent in the environment. The aim of this study was to assess the survival of Ad41 based on genome stability and infectivity in different types of manure and three types of biosolids. For viral survival studies, Ad41 was added to pelletized poultry litter (PL), alum-treated poultry litter (AL), raw poultry litter (RPL), liquid dairy manure (DM), swine manure (SM), and three types of biosolids 1, 2, 3. All samples were stored at 20 or 4°C and analyzed every 10 days for up to 60 days. Quantification PCR (qPCR) standard curves were generated for PL, AL, biosolids 1, and DM to measure the number of viral genomic copies remaining in the samples. To study the infectivity, all contaminated manure/biosolids samples were added to mammalian cell culture and viral mRNA was detected using one-step RT–PCR. Overall, Ad41 viral genomes were stable at both 20 and 4°C and there was no significant loss of viral DNA after 60 days in PL, AL, biosolids type 1, and DM. However, infectivity was lost almost immediately in high pH biosolids type 2 and 3, and infectivity decreased quickly in DM, with estimated T90 of 4.3 and 8.7 days at 20 and 4°C, respectively. Ad41 had ~1.9 log loss of infectivity after added in SM and biosolids type 1 at day 0, and estimated T90 was 12.5 and 28.6 days for biosolids type 1, and 19.1 and 51.0 days for SM at 20 and 4°C, respectively. Ad41 maintained infectivity in all three poultry litter, and after 60 days incubation, there were significantly more infectious virus in PL, AL, and RPL than biosolids 1, SM, and DM at 20°C.     

医疗废物管理对环境影响的初探

医疗废物的传染性可能对人类和环境造成不良影响,因此医疗废物管理是非常重要的.首先概述了医疗废物处理在各国的立法情况,以及医疗废物的收集和分类情况,进一步讨论了焚烧和高压釜处理医疗废物方法的优缺点,焚烧会造成二恶英、呋喃和汞排放,造成不利的健康和环境影响,而高压釜处理不能处理所有类型的医疗废物.控制医疗废物的最佳手段是减少产生量,可以通过对医护人员进行良好的培训以及实施标准化医疗废物分类和收集来实现,并研究改进传染性医疗废物处理方法.