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,...     

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%).     

微囊藻毒素-RR对烟草细胞活力及营养生理的影响

采用0.1,1.0,10.0μg/mL的微囊藻毒素-RR(MC-RR)处理烟草BY-2悬浮细胞,测定了细胞活力、细胞内蛋白质含量、可溶性糖含量、硝态氮含量及总磷含量,并且检测了酸性磷酸酶(ACP)的活力变化情况.结果表明,中、高浓度毒素处理细胞2d后,细胞活力及蛋白质含量与对照相比均显著下降.高浓度MC-RR处理降低了胞内可溶性糖的含量,暴露2d后仅为对照的45.57%;低浓度MC-RR处理在后期增加了胞内可溶性糖含量.高浓度毒素处理细胞4d后,细胞内硝态氮含量显著低于对照;中、低浓度毒素处理细胞7d后降低了胞内硝态氮含量.3组毒素处理均降低了胞内总磷含量,到实验结束时,低、中、高浓度处理组的胞内磷含量分别为对照的74.98%、76.47%和84.00%.3组处理组ACP活力与对照相比呈现先降低后升高的趋势.     

大气细颗粒物暴露干扰心肌细胞存活和炎性反应的主要毒性组分研究

鉴于PM_(2.5)组分呈现多样性和复杂性,其引起心力衰竭的主要毒性组分尚不清楚.由于采样地区、季节和污染源的不同,PM_(2.5)不同组分的占比也会有巨大差异,但不同浓度无法比较其不同组分间的毒性差异.因此,为了揭示大气PM_(2.5)引起心肌毒性作用的关键组分,分离复合型PM_(2.5)的3种主要组分(有机组分、金属组分和水溶性组分),综合两种浓度暴露方式,即实际占比浓度和与复合型PM_(2.5)相同的暴露浓度,染毒H9C2大鼠心肌细胞24 h和48 h,选择心肌细胞的细胞存活和炎症反应为主要评价指标,旨在揭示PM_(2.5)对心肌造成毒性损伤的关键性成分.CCK-8法检测存活结果显示,不同组分在实际环境比重下,对心肌细胞存活率没有造成显著影响.相同浓度暴露下,高剂量组分(30μg·cm~(-2))引起心肌细胞存活率显著降低,且有机组分毒性大于其他组分.根据细胞活力测定结果,选择低染毒浓度(10μg·cm~(-2))暴露细胞,采用qRT-PCR和ELISA试剂盒检测炎症因子变化.与对照组相比,金属组暴露后,炎症因子TNF-α和IL-1β显著升高,而有机组则显著升高TNF-α的含量.结果表明,造成心肌细胞存活毒性和炎症损伤的主要PM_(2.5)组分可能为有机组分和金属组分,而炎性反应对这两种组分的响应存在显著差异.     

Better understanding of the activated sludge process combining fluorescence-based methods and flow cytometry: A case study

This study aims to demonstrate the validity of fluorescence-based methods, together with flow cytometry, as a complementary tool to conventional physicochemical analyses carried out in wastewater treatment plants (WWTPs), for the control of the currently largely unknown activated sludge process. Staining with SYTO 9, propidium iodide and 5-(and 6)-carboxy-2′,7′-difluorodihydrofluorescein diacetate (carboxy-H₂DFFDA) was used for cell viability and oxidative stress monitoring of the bacterial population forming the activated sludge of a WWTP. Throughout the period of research, several unstable periods were detected, where the non-viable bacteria exceeded the 75% of the total bacterial population in the activated sludge, but only in one case the cells with oxidative stress grew to 9%, exceeding the typical values of 2%–5% of this plant. These periods coincided in two cases with high values of total suspended solids (SST) and chemical oxygen demand (COD) in the effluent, and with an excess of ammonia in other case. A correlation between flow cytometric and physicochemical data was found, which enabled to clarify the possible origin of each case of instability in the biological system. This experience supports the application of bacterial fluorescence staining, together with flow cytometric analysis, as a simple, rapid and reliable tool for the control and better understanding of the bacteria dynamics in a biological wastewater treatment process.     

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.     

改良MTT法在鱼类细胞毒性实验中的使用条件

文章利用斑点叉尾鮰鱼卵巢细胞(CCO)、鲤鱼上皮癌细胞(EPC)、草鱼肾细胞(CIK)探讨改良四噻唑蓝(MTT)比色法在测定污染物对鱼类细胞急性毒性实验中的最佳条件。通过实验发现其最佳实验条件为570nm波长下,MTT作用4h,MTT终浓度1mg/mL,最佳接种量的确定因每种细胞的特性而不同,生长速度快的细胞接种量较少。     

垃圾渗滤液与再生水生物毒性的体外检测方法应用

本文总结了近年来在垃圾渗滤液和再生水生物毒性检测方面的主要体外试验模型和检测方法,并整理了这些模型和方法在生物毒性评价中的应用.目前常用的体外试验模型包括人源细胞系、其他哺乳动物细胞以及微生物细胞,相比较而言,人源细胞系在检测结果外推至人体时具有更强的说服力,因而其应用最为广泛.体外检测方法可概括为细胞毒性、遗传毒性和内分泌干扰效应检测三个方面,其中内分泌干扰效应的研究较多集中于雌激素效应.最后,本文提出开发三维体外细胞模型、体外试验与化学分析相结合、全面分析内分泌干扰效应和建立成组体外试验体系是渗滤液和再生水生物毒性检测方法的发展方向.     

An investigation of methods for enriching trophoblast from maternal blood

Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD 16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.     

甲醛致DNA-蛋白质交联作用及其修复的研究

为了探讨甲醛致生物机体DNA-蛋白质交联作用及其修复能力,以昆明纯系小鼠和人肝癌细胞系HepG2为实验材料分别进行体内和体外实验,采用KCl-SDS沉淀法来检测甲醛染毒后小鼠肝细胞和HepG2细胞中DNA-蛋白质交联的含量及其修复效果.体内实验结果表明,低浓度的气态甲醛(0.5 mg·m-3)不能引起DNA-蛋白质的交联,较高浓度的甲醛(1.0 mg·m-3,3.0 mg·m-3,p＜0.01)可以产生明显的DNA-蛋白质交联作用;由3.0 mg·m-3浓度的气态甲醛产生的DNA-蛋白质交联在12h内可以得到明显的修复,并在24 h内恢复到空白对照水平.体外实验结果表明,经低浓度液态甲醛(25 μmol·L-1和50μmol·L-1)处理后,HepG2细胞内的DPC系数虽然稍有变化,但是与空白对照组相比较无显著差异,而当甲醛浓度上升至75 μmol·L-1及以上时,细胞中的DPC系数出现了极显著上升(p＜0.01);采用75 μmol·L-1甲醛染毒HepG2细胞,在染毒18h和24 h后,细胞内的DPC水平较染毒结束时发生了极显著的下降(p＜0.01),且与空白对照组相比无显著差异.以上结果显示,低浓度的甲醛不能引起DNA-蛋白质的交联,较高浓度的甲醛可以引起明显的DNA-蛋白质的交联作用,且甲醛所致的DNA-蛋白质交联在体内修复比体外修复所需时间要短.     

Detection of both the normal and mutant alleles in single cells of individuals heterozygous for the sickle cell mutation—prelude to preimplantation diagnosis

As a preliminary step to preimplantation diagnosis of sickle cell disease in unfertilized eggs or 8-cell embryos of heterozygous parents, we established quality control for detection of the mutant and normal alleles of the beta-haemoglobin gene using single buccal cells. Efficient polymerase chain reaction (PCR) amplification of a 680 base pair sequence of the beta-globin gene spanning the site of the sickle cell mutation was obtained for 79 per cent of single heterozygous cells. In 71 per cent of cases, both alleles were detected. With this current efficiency, we predict that a clinical preimplantation diagnosis at the 8-cell embryo stage could be carried out safely and reliably for a couple at risk of transmitting sickle cell disease to their children.     

Chorionic villus culture for prenatal diagnosis of chromosome defects: Reduction of the long-term cultivation time

We report in detail two series of chorionic villus cultivation for prenatal chromosomal diagnosis. Chorionic villi were sampled from both first- and second-trimester pregnancies. One hundred cultures were treated with trypsin–EDTA for 2 h and collagenase overnight, (method A) and 100 were treated with trypsin–EDTA for 1 h and collagenase for 2 h (method B). Using short-term enzymatic digestion, the cultivation time was reduced from 14 days to 6 days. Sufficient amounts of metaphases of good quality were present in 93 per cent of primary cultures harvested in situ, whereas enough metaphases of sufficiently good quality were in most cases present only after subcultivation of the cultures using method A. The decrease in cultivation time obtained is probably due to a higher yield of viable cells in monocellular suspension, an increased attachment efficiency, and a more rapid attachment of single cells (within 24 h).