密度梯度分离纯化/免疫荧光技检测饮用水中"两虫"

采用膜溶解、密度梯度分离纯化结合免疫荧光染色法对饮用水中的"两虫"(隐孢子虫和贾第鞭毛虫)进行定性与定量检测,以大幅降低检测成本,筒化操作.结果表明,使用Percoll-蔗糖介质可取得良好的分离纯化效果,其卵囊和孢囊初始回收率分别为56%和35%,远高于EPAl623方法的要求.利用该方法对河网型水源水及水厂滤后水进行了检测,发现10 L水源水中有1-3个隐孢子虫和0-2个贾第鞭毛虫,但在50 L滤后水中均未检出"两虫".     

上海市浦东自来水厂净水工艺对两虫去除效果的研究

采用免疫磁分离及荧光染色法（EPA1623法）对上海市浦东地区4家自来水厂的原水、出厂水、管网水中贾第鞭毛虫和隐孢子虫的分布状况进行了分析，并考察了水厂净水过程中不同操作单元对两虫的去除效果。研究表明：原水中存在贾第鞭毛虫与隐孢子虫，但其密度分别维持在0～8个／10L和0～6个／10L的低水平，而在出厂水与管网水中均未检出两虫；水厂的混凝、沉淀单元对两虫具有明显的去除效果。     

碳酸钙沉淀法检测混浊原水中“两虫”的研究

对于混浊水源水中"两虫"的检测,选定有效的浓缩富集及分离纯化方法尤为重要。滤囊/滤芯富集—免疫磁分离(IMS)组合方法对混浊水源水的预处理效果较好,但成本过高。为此,研究了碳酸钙沉淀法对某混浊河水中"两虫"的浓缩富集效果,并对浓缩后样品比较了密度梯度离心法和免疫磁分离法的分离纯化效果。结果发现,利用碳酸钙沉淀法可有效实现水中"两虫"的富集;从分离纯化效果来看,Percoll—蔗糖密度梯度离心方法对贾第鞭毛虫和隐孢子虫的平均回收率分别可达72.3%和39.5%,相对标准偏差分别为10.8%和11.9%;与国标方法中免疫磁分离方法的效果相当,但可显著降低分析成本。因此,碳酸钙沉淀—密度梯度离心组合技术用于混浊水源水中"两虫"的检测具有较好的应用前景。     

浅谈水环境中隐孢子虫和贾第鞭毛虫的检测方法

水环境中微生物贾第鞭毛虫和隐孢子虫是两种严重危害水质安全的致病性原生寄生虫，主要通过水和食物等传播疾病，人和动物感染“两虫”所患疾病分别称为贾第鞭毛虫病和隐孢子虫病。当前，人们逐渐认识到其对人类的危害不容忽视。因此研究适合我国广大供水企业采用可靠的“两虫”检测方法极其重要，对保障我国城市供水水质安全具有重要的意义。现就“两虫”在水环境中的检测方法予以综述。     

某市贾第鞭毛虫和隐孢子虫污染现状

利用免疫荧光分析技术对南方某市饮用水源水、自来水厂出水和污水处理厂进、出水中贾第鞭毛虫和隐孢子虫(两虫)的污染状况进行了调查,并就自来水厂常规处理工艺对两虫的去除特性进行了研究.结果显示:该市饮用水源水中的两虫密度分别为2～120个/100L和0～24个/100 L,自来水厂出水分别为0～12个/1000 L和0～8个/1000 L,污水处理厂进水中的两虫密度分别为7 200～18 300个/L和69～1 210个/L,二级处理出水的分别为6～153个/L和1～46个/L;混凝沉淀和过滤对两虫有较好的去除效果.     

高藻水中“两虫”检测的预处理方法研究

针对密度梯度分离纯化法用于高藻水中贾第鞭毛虫和隐孢子虫("两虫")检测存在的问题,以铜绿微囊藻配制的模拟水源水为对象,探讨了氯氧化和加压两种预处理方法对于改善"两虫"回收率的效果。研究发现,预氯化处理虽然能有效破坏藻细胞,仍会对后续的分离纯化及镜检过程带来较大的干扰,而且藻细胞在沉淀过程中会裹带"两虫",导致回收率下降;而通过加压处理破坏藻细胞气囊,可促进藻细胞的分离,使"两虫"回收率达到与免疫磁分离方法相当的效果,且操作简便,处理成本低,具有良好的应用前景。     

上海市区原水及自来水中两虫分布调查

对上海市区15座水厂的原水和自来水的调查表明,长江原水及黄浦江上游原水中均有贾第鞭毛虫和隐孢子虫存在,以贾第鞭毛虫为主,原水中两虫的总体污染程度较低;在所有自来水样品中均未发现两虫存在;根据调查结果提出了去除两虫的手段和预警监测方法.     

BAC滤池对浊度和颗粒数的控制研究

传统的贾第鞭毛虫和隐孢子虫（简称“两虫”）检测方法存在诸多不足，为此选用浊度和颗粒数作为“两虫”的替代指标，以对浊度和颗粒物的去除率来衡量生物活性炭（BAC）滤池对“两虫”的控制效果。试验结果表明：采用颗粒数表征滤后水水质比采用浊度更适宜。过滤初期颗粒数从峰值降到50个／mL以下所需的时间比浊度降到0．1NTU所需的时间多1h左右。正常过滤期间BAC滤池进水浊度一般在0．1NTU以下，经过BAC滤池处理后，浊度得到进一步降低，平均去除率为52．7％。炭层对浊度的去除率为56．4％，其出水浊度基本上都低于0．05NTU，而砂层对浊度不但没有去除能力，反而使出水浊度平均上升了约3．7％。炭层对颗粒物的平均去除率为33．3％，砂层对颗粒物的平均去除率为8．5％。     

再生水中隐孢子虫和贾第鞭毛虫的检测方法研究

隐孢子虫和贾第鞭毛虫（简称“两虫”）是两种严重危害水质安全的病原性原生动物,但国际上常用的两虫检测方法——1623方法存在工作量大、成本高、回收率低等缺点。通过考察1623方法中浓缩、分离两个主要环节的替代方案,发现膜过滤—洗脱是比较理想的两虫浓缩方式,在免疫磁性分离（IMS）环节中酸解离比热解离的效果好。根据以上研究成果,建立了回收率较高（对两虫的回收率分别超过了30％和40％）而成本较低（为原方法的55％左右）的再生水两虫密度检测方法。进一步的研究表明,浓缩是限制低浊水（浊度约1NTU）回收率的主要步骤,而IMS是限制高浊水（浊度约4NTU）回收率的主要步骤。     

澳门地区原水及海水中的病原虫调查

贾第鞭毛虫隐孢子虫是水源性流行病中常见的病原体，通常以孢囊或卵囊的形式存在于水中，常用消毒剂对其消毒效果较差，我国目前尚无饮用水的病原虫标准，有关病原虫在我国饮用水系统中存在情况的调查也未见报道，澳门自来水公司化验研究中心在已完全掌握了病原虫的检测方法后，对澳门地区供水系统及海水水样中的病原虫现状进行了调查，结果显示，在受到污染的原水中有病原虫检出。     

Development of a nested-PCR assay for the detection of Cryptosporidium parvum in finished water

A nested-PCR assay, incorporating an internal positive control, was developed for Cryptosporidium monitoring in finished water. This assay was capable of reproducibly detecting 8 oocysts in spiked-filtered water samples collected from 5 South Australian water treatment plants. The RT-PCR assay of Kaucner and Stinear (Appl. Environ. Microbiol. 64(5) (1998) 1743) was also evaluated for the detection of Cryptosporidium parvum. Initially, under our experimental conditions, a detection level of 27 oocysts was achieved for spiked reagent water samples. This level was improved to 5 oocysts by modification of the method. Untreated South Australian source waters concentrated by calcium carbonate flocculation were found to be highly inhibitory to the RT-PCR assay. Concentration of similar samples using Envirochek filters appeared to eliminate PCR inhibition. While both methods possessed similar sensitivities the nested-PCR assay was more reproducible, more cost effective, simpler to perform and could detect both viable and non-viable intact Cryptosporidium parvum oocysts, which is an important consideration for plant operators. These factors make the nested-PCR assay the method of choice for screening large numbers of potable water samples, where a reliable low level of detection is essential.     

Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody-antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1x10(6)-1x10(2)oocystsml(-1). The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.     

用PCR技术检测水中隐孢子虫

介绍了一种检测水中隐孢子虫的巢式PCR方法：根据Cryptosporidium parvum 18s rRNA基因序列选用了两对引物，特异性扩增了该基因可变区中1056bp和435bp目标片段。加标试验表明，该方法的检测限为10个卵囊。     

应用细胞感染技术检测水中活性隐孢子虫

根据活性隐孢子虫感染人结肠癌细胞（HCT-8）产生感染斑（族）的特点,利用体外细胞培养技术和免疫荧光分析技术建立了活性隐孢子虫的检测方法,并利用最大可能数统计原理对样品中的活性隐孢子虫进行了定量.     

水中隐孢子虫和贾第鞭毛虫的替代检测指标

美国国家环保局（USEPA）制定并发布的1623方法是目前国际上常用的水中隐孢子虫和贾第鞭毛虫标准检测方法，该方法存在成本高、回收率低等缺陷。各国研究者试图通过考察其他水质指标与两虫的相关关系来寻找两虫密度和活性的有效替代指标，以间接反映两虫在水体中的存在水平，其中包括浊度、颗粒计数等物理学指标以及细菌、细菌孢子、病毒、噬菌体等生物学指标。虽然还未得出具有普遍性的结论，但研究表明颗粒计数、好氧孢子等有可能成为有效的两虫替代指标。     

Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst

Cryptosporidium parvum and Cryptosporidium hominis are obligate enteric protozoan parasites which infect the gastrointestinal tract of animals and humans. The mechanism(s) by which these parasites cause gastrointestinal distress in their hosts is not well understood. The risk of waterborne transmission of Cryptosporidium is a serious global issue in drinking water safety. Oocysts from these organisms are extremely robust, prevalent in source water supplies and capable of surviving in the environment for extended periods of time. Resistance to conventional water treatment by chlorination, lack of correlation with biological indicator microorganisms and the absence of adequate methods to detect the presence of infectious oocysts necessitates the development of consistent and effective means of parasite removal from the water supply. Additional research into improving water treatment and sewage treatment practices is needed, particularly in testing the efficiency of ozone in oocyst inactivation. Timely and efficient detection of infectious C. parvum and C. hominis oocysts in environmental samples requires the development of rapid and sensitive techniques for the concentration, purification and detection of these parasites. A major factor confounding proper detection remains the inability to adequately and efficiently concentrate oocysts from environmental samples, while limiting the presence of extraneous materials. Molecular-based techniques are the most promising methods for the sensitive and accurate detection of C. parvum and C. hominis. With the availability of numerous target sequences, RT-PCR will likely emerge as an important method to assess oocyst viability. In addition, a multiplex PCR for the simultaneous detection of C. parvum, C. hominis and other waterborne pathogens such as Giardia lamblia would greatly benefit the water industry and protect human health.     

Prevalence of Cryptosporidium oocysts and giardia cysts in the drinking water supply in Japan

A one-year monitoring of Cryptosporidium oocysts and Giardia cysts was conducted at a water purification plant. A total of 13 samples of 50 L river source water and 26 samples of 2,000 L-filtered water, treated by coagulation flocculation, sedimentation and rapid filtration, were tested. Prior to conducting a survey of a water purification plant, we developed a method for concentrating Cryptosporidium oocysts from a large volume of raw or filtered water using a hollow fiber ultrafiltration (UF) membrane, and this procedure was adapted to survey a water purification plant. Cryptosporidium oocysts were detected in all of the 13 raw water samples. The geometric mean concentration was 40 oocysts 100 L. Giardia cysts were detected in 12 of 13 raw water samples (92%) and the geometric mean concentration was 17 cysts/100 L. Probability distributions of both Cryptosporidium oocyst and Giardia cyst concentration in raw water were nearly lognormal. In filtered water samples, Cryptosporidium oocysts were detected in 9 of the 26 samples (35%) with the geometric mean concentration of 1.2 oocysts /1,000 L and Giardia cysts in 3 samples (12%) with 0.8 cysts/1,000 L. The estimated log10 removal efficiency of Cryptosporidium oocysts and Giardia cysts by rapid-sand filtration was 2.47 and 2.53, respectively. Empty particles were removed at a higher log10 than intact oocysts and cysts. The efficiency of particle removal in the rapid sand filtration process tends to be reduced under cold-water conditions. Close management is necessary in the winter when the water temperature is low.     

Inactivation and potential repair of Cryptosporidium parvum following low- and medium-pressure ultraviolet irradiation

This study investigated the level of inactivation and the potential for Cryptosporidium parvum to repair following low doses (1 and 3mJ/cm(2)) of ultraviolet (UV) irradiation from both low- and medium-pressure UV lamps. Cryptosporidium parvum oocysts suspended in phosphate buffered saline were exposed to UV using a bench-scale collimated beam apparatus. Oocyst suspensions were incubated at 5 degrees C or 25 degrees C under light and dark conditions up to 120 h (5 days) following exposure to UV irradiation, to examine photoreactivation and dark repair potential, respectively. Cryptosporidium parvum infectivity was determined throughout the incubation period using an HCT-8 cell culture and an antibody staining procedure for detection. No detectable evidence of repair was observed after incubation under light or dark conditions following either LP or MP UV lamp irradiation.