一种高效提取焦化废水活性污泥总DNA的方法

对焦化废水活性污泥中微生物建立高质量的总DNA提取方法是开展分子生态学研究的重要前提.通过反复冻融-蛋白酶K-SDS、溶菌酶-反复冻融-SDS及溶菌酶-反复冻融-蛋白酶K-SDS这3种综合方法对焦化废水活性污泥总DNA进行提取,以OD260/OD280、OD260/OD230、产率、片段完整性、片段大小5个指标来评价样品总DNA的提取效果.结果表明,溶菌酶-反复冻融-蛋白酶K-SDS法所提取的总DNA的OD260/OD280值约为1.8,产率为1.90～16.30 μg·g-1,片断完整性好,主带清晰,大小约为23 kb,其PCR反应抑制物少,能够直接进行16S rRNA基因的PCR扩增.溶菌酶-反复冻融-蛋白酶K-SDS法能够为焦化废水活性污泥中微生物的分子生态学研究提供高质量的总DNA.     

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,...     

用于分子生态学研究的堆肥DNA提取方法

分子生态学为堆肥微生物的研究提供了新的技术手段,DNA的提取是该技术的基础,但由于腐殖酸类物质的污染,增加了堆肥微生物总DNA的提取难度.采用了3种不同的方法(溶菌酶法、超声波破碎法和蛋白酶K-CTAB法)从堆肥中提取微生物的总DNA,使用核酸和蛋白质分析仪检测后表明3种提取方法获得的DNA产量均较高;琼脂糖凝胶电泳结果表明其长度约为23 kb;使用细菌16S rRNA基因通用引物(27F和1 495R)对总DNA进行PCR扩增,都获得了几乎全长的16S rDNA序列(约1.5 kb);利用限制性内切酶(Hae Ⅲ和AluⅠ)对纯化后的PCR产物进行RFLP分析,结果表明3种方法提取的DNA反映了比较一致的微生物多样性.虽然3种方法各有优缺点,但其提取的DNA都可以用于堆肥微生物的分子生态学研究,可以根据实际需要选用某一种方法用于提取堆肥总DNA.     

大连太平洋牡蛎体内寄生虫:沿岸单孢子虫检测

于2007年3月,采集大连大窑湾浮筏贝类养殖区的太平洋牡蛎,抽取牡蛎的血淋巴液进行沿岸单孢子虫的研究。采用显微镜观察、聚合酶链式反应(PCR)和原位杂交(ISH)方法对血淋巴液样品进行检测。结果在显微镜下观察到牡蛎血淋巴液中存在沿岸单孢子虫(Haplosporidium costale,SSO)原生质体的类似物;提取血淋巴液基因组DNA,应用扩增沿岸单孢子虫SSUrDNA区域的引物对进行PCR扩增,产生约150bp的基因片段,经测序并将测序结果与基因库中已知序列比对分析,确定这种类似物为沿岸单孢子虫。同时,采用沿岸单孢子虫的特异性DNA探针SSO1318,对牡蛎血淋巴液中的沿岸单孢子虫进行原位杂交,结果为阳性。     

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.      

海洋沉积物中细菌DNA和RNA水平群落差异

海洋沉积物中的微生物在生物地球化学循环中具有重要作用.目前,细菌群落的研究多基于16S rRNA基因(DNA)展开,但DNA不仅包括活性微生物DNA也包括非活性微生物DNA,而RNA水平则可表征环境中具有活性的微生物种群.本研究采用荧光定量PCR和Illumina高通量测序技术,在DNA和RNA水平上研究了渤海和南黄海表层沉积物中细菌群落结构.结果表明,细菌DNA基因丰度比RNA基因丰度高1～2个数量级,DNA水平群落多样性高于RNA水平,二者群落结构差异显著.沉积物中的细菌具有活跃的化能异养、硫酸盐还原和硝化作用.基于16S rRNA基因的测序在探索微生物群落功能时可能会误判重要的功能微生物,总细菌群落中的"稀有生物圈"可能包含转录活跃者,发挥着重要的生物地球化学作用.总体而言,在分析来自稳定沉积环境的细菌群落结构时,基于16S rRNA的研究更能反映真实的生态状况.     

空调冷却塔水与空气军团菌的PCR方法快速检测

应用 Enviro Amp PCR-反向杂交法(涉及 5S rRNA基因和 mip基因 )和半巢式 PCR(涉及 16S rRNA基因 )成功检测人工气溶胶化的军团菌 ,这些方法进一步用于检测空调系统冷却塔的水及由其产生的气溶胶中的军团菌 ,从空气样品中检测到军团菌属及嗜肺军团菌 .检测的冷却塔水样品 100%含军团菌属细菌 ,其中 41.6%含嗜肺军团菌 .2种方法相比 ,半巢式 PCR法更经济、方便 .     

长牡蛎尼氏单孢子虫的初步研究

采集大连大窑湾养殖区的长牡蛎(Crassostrea gigas),抽取其血淋巴液在显微镜下观察,观察到尼氏单孢子虫(Hap-losporidium nelsoni)原生质体的类似物.对抽取的血淋巴液进行总基因组DNA的提取,将扩增单孢子虫(Haplosporidiosis)SSUrDNA区域的引物对HAP-F2和HAP-R2进行改进,经过特异性试验证明其特异性很好,得到引物对HAP-F和HAP-R,应用此对引物进行聚合酶链式反应(PCR),结果扩增出239bp条带.将PCR产物进行测序,经序列比对分析确定为尼氏单孢子虫.同时,采用尼氏单孢子虫的特异性DNA探针MSX1347对抽取的长牡蛎血淋巴液进行原位杂交,结果也检测到了尼氏单孢子虫.     

超声灭活饮用水中隐孢子虫研究

为研究超声对饮用水中隐孢子虫(Cryptosporidium parvum)的灭活情况,考察了超声频率、功率、pH值和温度对灭活率的影响,通过形态学观察初步探讨了超声灭活隐孢子虫的机制,并进行了灭活动力学分析.结果表明,低频有利于隐孢子虫灭活,19.8kHz,pH7.2,温度(20±1)℃条件下超声15min灭活率可达92.5%,频率升高灭活率反而下降.在本实验条件下,超声功率103W对隐孢子虫的灭活效果与151W的相近,pH值对超声灭活隐孢子虫的影响不大,36℃超声灭活15min灭活率为95.6%,而在9℃下超声15min灭活率为88.3%,水温升高有利于灭活.灭活前后的形态学变化表明超声空化作用导致细胞膜破坏,细胞质流出从而起到灭活孢囊的效果.超声灭活隐孢子虫遵循假一级反应动力学,灭活隐孢子虫以低频率高功率的效果最好,可认为隐孢子虫的灭活以超声空化的强度为主.     

城市污水再生处理过程中病原性原虫的去除特性

通过定期监测北京市某城市污水再生处理过程各单元出水中的隐孢子虫和贾第鞭毛虫(两虫)浓度,系统考察了污水再生处理系统对两虫的去除特性.结果表明,原污水、初沉池出水、二沉池出水、絮凝沉淀池出水和砂滤池出水中隐孢子虫的平均检出量分别为238、179、6、1、0.3个·L-1,贾第鞭毛虫的平均检出量分别为1 568、1048、22、4、0.6个·L-1.污水再生处理系统对隐孢子虫和贾第鞭毛虫的总去除率分别为2.98log(99.895%)和3.46log(99.965%).一级处理工艺对污水中两虫的去除效果并不理想,去除率分别只有0.13log和0.18log.二级生物处理对两虫的去除贡献最大,去除率分别达1.50log和1.67log.污水深度处理工艺(絮凝-沉淀-砂滤)能有效提高两虫的去除效果.污水厂进水中的两虫检出量随季节变化,雨季较低,旱季较高.     

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%).     

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]     

上海水源水库产2-MIB生物总量评估方法与应用

本研究开展了蓝藻产2-MIB功能基因mic的qPCR定量分析,对上海水源水库中的mic基因进行长期调研.结果表明,在长江水源水库和黄埔江水源水库中mic基因都有检出.在夏季其丰度达到峰期,基因拷贝数达到10~7～10~8 copies·L~(-1).分析表明mic基因拷贝数与蓝藻、伪鱼腥藻细胞密度、2-MIB浓度之间均存在相关性.因此通过mic基因定量进而推断水中产嗅生物总量的方法,可代替繁琐、重现性低的显微镜镜检过程.应用中还需更多实践,结合其他指标改进准确度.     

Occurrence of Viruses and Protozoa in Drinking Water Sources of Japan and Their Relationship to Indicator Microorganisms

A nationwide survey of viruses, protozoa, and indicator microorganisms in drinking water sources of Japan was conducted. Among 64 surface water samples collected from 16 drinking water treatment plants, 51 (80?%) samples were positive for at least one of the 11 pathogen types tested, including noroviruses of genogroups I (positive rate, 13?%) and II (2?%), human sapoviruses (5?%), human adenoviruses of serotypes 40 and 41 (39?%), Cryptosporidium oocysts (41?%), and Giardia cysts (36?%). Total coliforms, Escherichia coli, and F-specific coliphages were detected in 63 (98?%), 33 (52?%), and 17 (27?%) samples, respectively, and E. coli was judged to be the most suitable indicator of pathogen contamination of drinking water sources. Genogroup-specific real-time PCR for F-specific coliphages revealed the presence of F-specific RNA coliphages of animal genogroup I and human genogroups II and III in 13 (41?%), 12 (39?%), and 1 (3?%), respectively, of 31 plaques isolated.     

高铁酸盐对藻类肝毒素的降解

研究高铁酸盐对悦目颤藻(Oscillatoria amoena)肝毒素(Microcystin-LR)的降解效能及其与pH的关系. 结果表明,处理有机质含量很高的藻类肝毒素粗提液,当高铁投加量增到40mg/L,pH控制在6～10,肝毒素几乎被完全降解.同时高铁的还原产物Fe³⁺、Fe(OH)₃发挥其助凝、絮凝的作用,对水体中有机质吸附沉降去除,TOC去除率达到50%左右,铁几乎无残留.高效液相色谱分析发现,作用机制可能是高铁氧化或异构化Adda基团的共轭双键,使Adda基团的结构发生变化,从而降低其毒性.     

Risk assessment of Giardia in rivers of southern China based on continuous monitoring

The occurrence and risks of Giardia in China have been unclear to date, which has made it difficult to properly manage source water as well as to create reasonable drinking water standards. The levels of Giardia in river networks of several cities in Zhejiang Province, China were found to be in the range of 0-5 oocysts/10 L in the rainy season in 2008. The mortality due to Giardia infection for people in this region was calculated to be from 0 to 1.95 × 10^-8 persons using a conditional probability equation. Based on multiple unboiled water intake routes, the disability-adjusted life years (DALYs) due to Giardia infection for people who consumed conventionally treated water was 0.625 (95% CI: 0.137-2.05) per 10⁵ persons, with the symptom of hospitalization making the highest contribution to total DALYs (0.56 per 10⁵ persons; 95% CI: 0.122-1.84). The DALYs decreased to 0.425 (95% CI: 0.137-2.05) per 10⁵ persons per year for those consuming water treated with advanced technology. These values were lower than the acceptable risk (1.97 × 10^-5 DALYs per year). This study revealed the risk of Giardia infection to the people in river networks of Zhejiang Province for the first time, and provides a method to evaluate the risk of Giardia infection. The results are useful for the modification of drinking water quality standards based on cost-benefit analysis.     

Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR)

The detection of viable bacteria in wastewater treatment plants(WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide(PMA) combined with the quantitative polymerase chain reaction(PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 μmol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.     

华东地区某饮用水源地中磺胺类抗性基因的分布特征

饮用水源中检测到的抗生素抗性基因(antibiotic resistance genes,ARGs)对饮用水质安全和人体健康产生的潜在威胁受到广泛关注.在掌握了华东地区某饮用水源地13种磺胺类抗生素的污染特征基础上,进一步采用定性PCR和荧光定量PCR解析该饮用水源水和底泥中磺胺类ARGs(sul1、sul2)以及抗性基因可转移元件Ⅰ型整合酶基因(int I1)的分布特征.结果表明,3种基因在该饮用水源水和底泥中均100%检出,sul1基因是该饮用水源地中检出含量最高的磺胺类ARGs,在水源水中含量范围为1.5×104~6.4×105copies·mL~(-1),底泥中则高达1.6×108copies·g~(-1),较sul2、int I1基因分别高0.6~2.2、0.5~1.9个数量级.sul1、sul2和int I1基因在该水源地入水口和出水口处的绝对含量无显著差别,而在底泥中sul1、sul2和int I1基因的绝对含量则是出水口高于入水口.sul1在夏季水源地出水口的检出含量最高,为6.4×105copies·mL~(-1);int I1基因在冬季的检出含量高于其他季节.sul1基因与13种磺胺类抗生素具有相关性(r=0.69,P0.05),其中与磺胺甲唑的含量显著相关(r=0.79,P0.01);int I1与sul1、sul2的相对含量之间也存在正相关关系(r为0.80和0.73,P0.05),这表明int I1在磺胺类ARGs的水平转移过程中起到了重要作用.本研究为典型饮用水源地中ARGs的污染现状提供基础数据,也为管控饮用水环境的抗性基因污染和制定管理决策提供依据.     

应用FCM-qPCR方法定量检测水中常见病原体

以往对水体病原体的研究主要是通过监测粪大肠杆菌作为指示,然而研究表明粪大肠杆菌与水中病毒和细菌病原体呈现出较差的相关性.因此,选取水中典型病原体并对其进行定量检测是当前需要解决的技术问题.为此本研究建立了流式细胞术和定量PCR联合使用方法,用于快速获取水环境中总病毒、细菌以及几种典型病原体(大肠杆菌、军团菌、腺病毒、贾第虫和隐孢子虫等)的浓度水平,并将该方法应用到污水处理厂进出水及受纳河流上下游的病原体检测中.结果表明,该污水处理厂对总细菌和总病毒以及几种典型病原体都具有较高的去除率(93%);污水处理厂排水对受纳水体病原体浓度水平基本没有负面影响.研究为评估污水处理厂处理效果及排水对受纳水体的生态影响提供了技术支持.