通用引物PCR方法在地表水病原菌检测中的应用

为了探索通用引物PCR方法在地表水病原细菌检测中的应用价值,利用16S rRNA基因的高度保守性,设计并合成细菌的通用引物,采用合成的引物扩增参考菌株及西安市区不同地表水样,并对PCR产物分别进行序列测定及序列同源性分析,同时检测水样中的细菌总数和粪大肠杆菌浓度.结果显示,通用引物扩增4种参考菌株,320 bp处均可得到清晰的电泳条带,其特异性通过序列测定及同源性分析得到进一步验证;通用引物PCR可检出250 ng/L的埃希氏大肠杆菌标准菌株DNA,其PCR检测的灵敏度可达0.275 CFU/mL;用通用引物对西安市区不同地表水样进行PCR扩增,其中■河、兴庆湖、大唐芙蓉园北湖、北石桥污水处理厂出水样品320 bp处都得到了清晰的电泳条带,而黑河水样没有条带;与之相对应的粪大肠杆菌群检测结果显示,只有北石桥污水处理厂出水和兴庆湖水样有粪大肠杆菌检出.      

聚合酶联反应检测医院处理后污水中的结核杆菌DNA片段

1材料和方法1．1水样来源为徐州市某综合医院污水处理后水样，以不同日期，经无菌处理后，用一次性容器取样20ml，共取10份水样。对照组用无菌蒸馏水。1．2抽提方法1．2．1水样标本DNA抽提：水样中结核杆菌DNA抽提按文献报道操作。1．2．2盲法对照试验中结核杆菌管DNA抽提：取1．5mlEppendrof管20只，前16管中加生理盐水400微升，另4管加无菌蒸馏水400微升。前16管随机在三管中各加入20个结核杆菌及对照细菌（结核杆菌以外常见细菌），实验人员事先不知各管细菌种类。DNA抽提方法同前。1．2．3聚合酶联反应（PCR）技术：引物由第四军…     

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.      

一般性问题

XSOZ 9903418用PCR技术检测生物硝化池污水中硝化细菌(Nitrobacteria)的研究/明镇寰…(浙江大学西溪校区生命科学学院)//浙江大学学报(理学版)/浙江大学一1999,26(2)一83一86 环信N一27 从生物硝化池污水水样中分离了硝化细菌DNA,以其为模板,针对其16SrRNA基因及23SrRNA基因而设计合成了两对引物并分别进行了PCR扩增,扩增产物的2%琼脂糖凝胶电泳结果显示,硝化细菌DNA模板得到了特异性扩增。参照分子量标准(Marker)的电泳结果,被扩增的DNA条带大小分别约为4oobp和73obp。实验结果表明PCR技术可用于污水中硝化细菌的快速检测。…     

城市污水二级处理出水中沙门氏菌的PCR检测

建立了一种利用PCR检测城市污水二级处理出水中沙门氏菌的方法.采用膜吸附-洗脱法浓缩二级出水水样中的沙门氏菌,通过选用一对涵盖性和特异性均较强的引物139和141,对浓缩后的沙门氏菌进行PCR检测.利用正交试验法,对沙门氏菌PCR体系进行五因素四水平的试验,建立了沙门氏菌PCR的最佳体系.通过梯度PCR考察了退火温度对检测结果的影响,选择65℃作为退火温度,优化后沙门氏菌PCR体系的检测灵敏度可达0.314ngL.通过对已知细菌浓度的水样进行检测,确定了该法检测水样中沙门氏菌的灵敏度为2130CFUL.与传统MPN法的相比,该法在检测效果和检测效率上都优于MPN法.     

生物硝化池污水中硝化细菌的快速定量研究

实验采用聚合酶链式反应（PCR）技术与最大几率数法（MPN）相结合的MPN－PCR法对生物硝化池污水中的硝化细菌进行快速定量。所用的一对PCR引物是在对硝化细菌的16SrRNA基因进行系统比较的基础上设计合成的，可以扩增出大小为388bp的DNA片段。以从生物硝化池污水中抽提的含硝化细菌DNA的混合DNA为模板，进行PCR扩增并确定合适的扩增条件。运用MPN－PCR法进行定量检测的整个过程可在几小时之内完成。     

Quantitative Detection of Virulence Genes eaeA and rfbE of Pathogenic Escherichia coli in Municipal Wastewater by Real-Time PCR

针对病原性大肠埃希氏菌EHEC和EPEC的毒力基因eaeA和rfbE,选用特异性引物,建立了实时荧光定量PCR检测方法.利用从污水中分离出的目的核酸片段构建重组质粒,通过测序和BLAST比对分析,确定了PCR扩增的特异性.将重组质粒作为模板,分别测定标准曲线,在eaeA基因模板量8.77～8.77×105 copy,rfbE基因模板量4.98 ～4.98×105copy的范围内,Ct(循环阈值)与模板量的对数值具有良好的线性关系[ R2为0.997( eaeA)和1.000(rfbE)].结合膜吸附洗脱浓缩方法,对于实际水样,eaeA和rfbE基因的检测限分别为1.75×102和9.96×101copy/100 mL.利用该方法对城市污水处理厂进、出水进行检测的结果显示,污水二级处理工艺对这2种毒力基因的去除效果明显.     

污水的青海弧菌Q67生物毒性检测及影响因素分析

应用淡水发光细菌--青海弧菌Q67对氧化沟处理工艺各处理单元进出水的生物急性毒性进行检测,对污水急性毒性检测的影响因素进行了分析.结果表明,水样取回后立即进行发光细菌毒性实验,水样均显示为刺激发光效应,污水毒性被掩盖;将水样放置1 d后进行毒性实验,部分水样显现出抑制发光效应;将污水用0.45μm微滤膜过滤可以使水样的...     

城市污水中病原性大肠埃希氏菌毒力基因eaeA和rfbE的实时荧光定量PCR检测

针对病原性大肠埃希氏菌EHEC和EPEC的毒力基因eaeA和rfbE,选用特异性引物,建立了实时荧光定量PCR检测方法. 利用从污水中分离出的目的核酸片段构建重组质粒,通过测序和BLAST比对分析,确定了PCR扩增的特异性. 将重组质粒作为模板,分别测定标准曲线,在eaeA基因模板量8.77～8.77×105 copy,rfbE基因模板量4.98～4.98×105copy的范围内,Ct(循环阈值)与模板量的对数值具有良好的线性关系〔R2为0.997(eaeA)和1.000(rfbE)〕. 结合膜吸附洗脱浓缩方法,对于实际水样,eaeA和rfbE基因的检测限分别为1.75×102和9.96×101copy/100 mL. 利用该方法对城市污水处理厂进、出水进行检测的结果显示,污水二级处理工艺对这2种毒力基因的去除效果明显.      

饮水中洋葱伯克霍尔德菌的分离鉴定及消毒剂灭活试验

依据GB/T 14926.41—2001《实验动物无菌动物生活环境及粪便样本的检测方法》从无菌环境中无菌操作取水样进行检测时,分离到一种粗短革兰氏阴性杆菌,进一步进行病原菌培养特性、生化鉴定和16S rRNA通用引物PCR扩增分析,结果为洋葱伯克霍尔德菌;并选用常用消毒剂对其进行悬液定量杀菌试验,84消毒液和75%乙醇对其作用10 min杀灭对数值≥5,50%甲酚皂溶液、新洁尔灭消毒液和10%癸甲溴铵溶液对其作用10 min杀灭对数值均5。     

Effects of two sludge application on fractionation and phytotoxicity of zinc and copper in the soil

The potential harm of heavy metals is a primary concern in application of sludge to the agricultural land. A pot experiment was conducted to evaluate the effect of two sludges on fractionation of Zn and Cu in soil and their phytotoxicity to pakchoi. The loamy soil was mixed with 0%, 20%, 40%, 60% and 80% (by weight) of digested sewage sludge (SS) and composted sludge (SC). The additions of both sludges caused a significant raise in all fractions, resulting in that exchangeable (EXCH) and organic bound (OM) became predominance of Zn and organic bound Cu occupied the largest portion. There was more available amount of Zn and Cu in SS treatments than SC treatments. During the pot experiment, the concentration of Zn in EXCH, carbonate (CAR) and OM and Cu in EXCH and OM fractions decreased in all treatments, so their bioavailability reduced. Germination rate and plant biomass decreased when the addition rate was high and the best yield appeared in 20% mixtures at the harvest of pakchoi. The two sludges increased tissue contents of Zn and Cu especially in the SS treatments. Zn in pakchoi was not only in relationship to ΔEXCH and ΔCAR forms but also in ΔOM forms in the sludge-soil mixtures. Tissue content of Cu in pakchoi grown on SC-soils could not be predicted by ΔEXCH. These correlation rates between Zn and Cu accumulation in pakchoi and variation of different fractions increased with time, which might indicate that sludges represented stronger impacts on the plant in long-term land application.     

Intraspecific differences in effects of co-contamination of cadmium and

A hydroponic experiment was carried out to study intraspecific differences in the effects of different concentrations of cadmium (Cd)(0-10 mg/L) and arsenate (As(V)) (0-8 mg/L) on the growth parameters and accumulation of Cd and As in six wheat varieties Jing-9428, Duokang-1, Jingdong-11, Jing-411, Jingdong-8 and Zhongmai-8. The endpoints of wheat seedlings, including seed germination,biomass, root length and shoot height, decreased with increasing the Cd and As concentrations. Significant differences in seed germination, biomass, root length, shoot height and the accumulation of Cd and As were observed between the treatments and among the varieties (p ＜ 0.05). The lethal dosage 50% were about 20, 80, 60, 60, 80 and 20 mg As/L for Jing-9428, Duokang-1, Jingdong-11,Jing-411, Jingdong-8 and Zhongmai-8, respectively, and the corresponding values for Cd were about 30, 80, 20, 40, 60 and 10 mg Cd/L, respectively. Among the six varieties, Duokang-1 was found to be the most resistant to Cd and As toxicity, and Zhongmai-8 was the most sensitive to Cd and As co-contamination. The resistance of the six varieties was found dependant on the seedling uptake of Cd and As. Duokang-1 was the most suitable for cultivation in Cd and As co-contaminated soils.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

EDTA-enhanced phytoremediation of lead contaminated soil by Bidens maximowicziana

Phytoremediation is a potential cleanup technology for the removal of heavy metals from contaminated soils.Bidens maximowicziana is a new Pb hyperaccumulator,which not only has remarkable tolerance to Pb but also extraordinary accumulation capacity for Pb.The maximum Pb concentration was 1509.3 mg/kg in roots and 2164.7 mg/kg in overground tissues.The Pb distribution order in the B. maximowicziana was:leaf>stem>root.The effect of amendments on phytoremediation was also studied.The mobility of soil Pb and the Pb concentrations in plants were both increased by EDTA application.Compared with CK(control check),EDTA application promoted translocation of Pb to overground parts of the plant.The Pb concentrations in overground parts of plants was increased from 24.23-680.56 mg/kg to 29.07-1905.57 mg/kg.This research demonstrated that B.maximowicziana appeared to be suitable for phytoremediation of Pb contaminated soil,especially,combination with EDTA.     

Distribution and ecological effect of mercury in Laogang landfill, Shanghai, China

Laogang landfill near Shanghai is the largest landfill in China, and receives about 10000 t of daily garbage per day, Samples of topsoil and plants were analyzed to evaluate mercury pollution from the landfill. For topsoil samples, there were significant correlations among total mercury （HgT）, combinative mercury （Hgc） and gaseous mercury （HgG）, and content of total organic carbon （TOC）, but, no significantly relationship was found between Hg content and filling time. Hg content changes in vertical profiles with time showed that the average Hgv of profiles 1992, 1996, and 2000 was similar, but their average HgG was quite different. HgT was significantly correlated with Hgc in profile 1992 and 2000, and Hgv was significantly correlated with Hg6 in profile 1996. HgG/Hgv ratio in profile samples decreased in the order of （HgG,/HgT）1992〉（HgG/HgT）1996〉〉（HgG/HgT）2000. A simple outline of Hg release in landfill could be drawn： with increasing of filling time, degradation undergoes different biodegradation, accordingly, gaseous mercury goes through small, more, and small proportion to total mercury. Distribution of Hg in plants was inhomogeneous, following the order of leaf〉root〉stem. The highest value of leaf may be associated with higher atmospheric Hg from landfill. Ligneous plants （e.g. Phyllostachys glanca, Prunus salicina and Ligustrum lucidum） are capable of enriching more Hg than herbaceous plants.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Enzymatic oxidation of aqueous pentachlorophenol

The influence of the nonionic surfactant Tween 80 on pentachlorophenol （PCP） oxidation catalyzed by horseradish peroxidase was studied. The surfactant was tested at concentrations below and above its critical micelle concentration （CMC）. Enhancement of PCP removal was observed at sub-CMCs. The presence of Tween 80 in the reaction mixture reduced enzyme inactivation which occurred through a combination of free radical attack and sorption by precipitated products. A simple first-order model was able to simulate time profiles for enzyme inactivation in the presence or absence of Tween 80. At supra-CMCs, the surfactant caused noticeable reductions in PCP removal, presumably through micelle partitioning of PCP which precluded the hydrophobic PCP molecule from interacting with the enzyme.     

Decomposition of alachlor by ozonation and its mechanism

Decomposition and corresponding mechanism of alachlor, an endocrine disruptor in water by ozonation were investigated. Results showed that alachlor could not be completely mineralized by ozone alone. Many intermediates and final products were formed during the process, including aromatic compounds, aliphatic carboxylic acids, and inorganic ions. In evoluting these products, some of them with weak polarity were qualitatively identified by GC-MS. The information of inorganic ions suggested that the dechlorination was the first and the fastest step in the ozonation of alachlor.     

基于综合水质标识指数和对应分析法的校园河道水质评价

以三峡大学的校园河道求索溪为研究对象,利用综合水质标识指数法确定求索溪水质类别,分析其水质时空变化规律,并利用对应分析法得出求索溪中不同监测点的主要污染因子.研究结果表明:求索溪整体的综合水质标识指数为7.423,整体水质为劣V类(地表水环境质量标准GB 3838-2002)且黑臭.从时间变化来看,求索溪4月份的水质最差,5月份次之,4、5月份所有监测点的水质都劣于V类且黑臭;8月份水质最好,水质为Ⅳ类;从空间分布来看,8个监测点综合水质标识指数均超过6.0,水质为劣V类,其中6号监测点的水质相对最好,监测点3号的水质相对最差;对应分析法得出求索溪的整体水体污染程度受总氮因子的影响最大,其次为总磷.该研究拟为求索溪及类似校园河道的水环境治理研究提供基础依据和参考.     

Potential of plant polyphenol oxidases in the decolorization and removal of textile and non-textile dyes

In this study an effort has been made to use plant polyphenol oxidases; potato （Solanum tuberosum） and brinjal （Solanum melongena）, for the treatment of various important dyes used in textile and other industries. The ammonium sulphate fractionated enzyme preparations were used to treat a number of dyes under various experimental conditions. Majority of the treated dyes were maximally decolorized at pH 3.0. Some of the dyes were quickly decolorized whereas others were marginally decolorized. The initial first hour was sufficient for the maximum decolorization of dyes. The rate of decolorization was quite slow on long treatment of dyes. Enhancement in the dye decolorization was noticed on increasing the concentration of enzymes. The complex mixtures of dyes were treated with both preparations of polyphenol oxidases in the buffers of varying pH values. Potato polyphenol oxidase was significantly more effective in decolorizing the dyes to higher extent as compared to the enzyme obtained from brinjal polyphenol oxidase. Decolorization of dyes and their mixtures, followed by the formation of an insoluble precipitate, which could be easily removed simply by centrifugation.