基于gyrB基因的地衣芽孢杆菌PCR快速检测方法的建立

根据地衣芽孢杆菌的gyrB基因序列设计一对引物,扩增片段大小为507bp的基因片段,该方法对地衣芽孢杆菌DNA的扩增结果为阳性,对照菌株扩增结果均为阴性,对地衣芽孢杆菌检测的灵敏性为1pg总DNA量,成功建立了地衣芽孢杆菌PCR检测方法,该方法具有快速、灵敏度高、特异性强等优点,为地衣芽孢杆菌的分离及鉴定奠定了良好的基础。     

STGC3基因启动子生物信息学分析及载体构建

目的运用生物信息学预测并分析鼻咽癌抑癌基因STGC3启动子,构建双荧光素酶报告基因表达载体。方法采用生物信息学软件预测STGC3基因5'端上游调控区5 000 bp序列进行启动子预测与分析,克隆最有可能的启动子,构建萤火虫荧光素酶双报告基因重组质粒,重组质粒经MluⅠ和BglⅡ限制性内切酶酶切及测序鉴定。结果 STGC3基因5'端上游-3 046 bp至-46 bp区域内有启动子活性,在-2 992 bp至-69 bp间启动子分值达0.8以上,其中-845 bp至-795 bp间分值最高,达到1.0。在-1 140 bp和-774 bp处检测到GC盒信号;在-441 bp处检测到CAAT盒信号。在-1 805 bp至-1 705 bp和-900 bp至-684 bp间各有一个Cp G岛;在-2 348 bp和-948 bp处各有一个转录起始位点。经不同长度的片段缺失比对,取最有可能的283 bp(-1 360 bp至-1 077 bp)、281 bp(-934 bp至-653 bp)和571 bp(-500 bp至+72 bp)启动子片段构建p GL3-en283、p GL3-en281及p GL3-en571三种质粒,质粒经酶切测序,酶切片段大小一致,序列正确。结论根据生物物信息学分析结果,成功构建STGC3基因启动子萤火虫荧光素酶报告基因表达载体,为双荧光素酶报告基因检测系统研究该基因启动子活性提供前期基础。     

食品中金黄色葡萄球菌PCR检测方法的建立

目的:建立快速检测食品中金黄色葡萄球菌的PCR方法。方法:优化金黄色葡萄球菌DNA提取方法并设计基因特异性引物,采用PCR方法扩增金黄色葡萄球菌的耐热核酸酶(nuc)编码基因,验证检测的特异性及灵敏度。结论:所检出的金黄色葡萄球菌在497bp处出现特异片段,该方法具有较高的敏感性和特异性,可作为食品中金黄色葡萄球菌快速检测的手段。     

TGEV和PEDV的多重PCR方法在猪传染性胃肠炎病毒检测中的应用研究

目的:通过应用同时检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)的多重RT-PCR方法,并对其进行特异性与敏感性试验。方法:对本地区150份临床疑似病毒性腹泻病料进行检测,检测了仔猪腹泻的粪便或小肠组织共150份病料。在实验室检测中,此方法能检测约50 TCID50的混合病毒,能够扩增出3条长度为750 bp(PEDV)、544 bp(TGEV)、275 bp(PARV)特异性片段,而其它病毒无特异性条带。结果:PEDV、TGEV阳性率分别为54.67%(82/150)、8.0%(12/150)。PEDV与TGEV混合感染率为5.33%。结论:同时检测猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)的多重RT-PCR方法的应用对临床上进行这两种疾病混合感染的检测具有重要意义。     

数字水准仪检定方法探讨

针对目前数字水准仪还没有形成一套完整有效的检测鉴定方法这一现状，分析了数字水准仪的原理和特性，提出了一套数字水准仪的系统检测鉴定方法。并按该方法进行了实验，实验检测数据表明该方法是合理可行的。     

鼻咽癌基因治疗的实验研究

目的研究Survivin表达下调和p53过表达对鼻咽癌细胞CNE-2的靶向杀伤作用。方法扩增hTERT启动子不同区段,构建真核表达质粒pcDNA3.1-hTERT-Survivin,用于启动Survivin反义寡核苷酸的表达;扩增p53基因,克隆到pcDNA3.1质粒中,构建p53的过表达载体pcDNA3.1-p53;联合pcDNA3.1-hTERT-Survivin和pcDNA3.1-p53转染鼻咽癌细胞株CNE-2,RT-PCR和免疫印迹法检测p53和Survivin的表达情况,MTT法检测CNE-2细胞活率情况,Annexin V/PI双染色法检测共转后的细胞凋亡情况。结果成功扩增270 bp和728 bp 2种不同大小片断的hTERT启动子区域,并与CMV增强子连接,以GFP为报告基因检测发现,270 bp启动子区段能较有效启动基因的表达;进一步成功构建Survivin反义寡核苷酸表达质粒pcDNA3.1-hTERT-Survivin和p53过表达载体pcDNA3.1-p53;转染鼻咽癌细胞株CNE-2后检测表明Survivin表达下调和p53表达上调;MTT和流式细胞检测显示联合转染pcDNA3.1-hTERT-Survivin和pcDNA3.1-p53可诱导鼻咽癌细胞株CNE-2的凋亡。结论利用hTERT启动子和CMV增强子可有效启动Survivin反义寡核苷酸的表达转染鼻咽癌细胞CNE-2后可抑制Survivin的表达,同时联合过表达p53基因后,可以有效诱导鼻咽癌细胞CNE-2的凋亡,提示该方法可以成为有效治疗鼻咽癌的一种新的靶向治疗方法。     

新型消毒副产物N-亚硝基二甲胺的研究进展

对N-亚硝基二甲胺（NDMA）的物化特性、水体中的来源、分析检测方法以及去除与控制工艺等研究进展进行了全面综述，并提出了未来的研究方向。由于NDMA特殊的物化性质，对该物质的检测和处理都有相当的难度。目前传统的检测方法还不能满足检测要求，需要建立快速、准确、可检测较低浓度NDMA的新型检测方法。对NDMA的去除主要是采用紫外线辐射处理工艺，但工程实践证明该方法性价比不高，因此，新型经济、有效的去除工艺及控制方法亟待研发。     

建筑结构检测鉴定方法探讨

根据多年建筑结构质量及安全性检测鉴定工作的经验，总结出从接受委托到最终做出鉴定报告的全过程方法，该方法在工程实践中取得了良好效果，本文介绍了该方法的主要内容，并结合工程实例，着重阐述了建筑结构质量及安全性调查，检测，分析，鉴定过程中的工作方法及要点。     

热流计法在建筑节能现场检测中的应用

热流计法是目前建筑节能检测领域常用的一种方法，简要介绍了热流计法的检测原理及相应的检测设备，并结合现场检测经验和相关标准提出一些使用要点，为该方法的正确使用提供参考。     

八尺门特大桥Ф1．5m钻孔嵌岩桩的静载试验

结合具体工程实践，对其工程地质状况作了分析，介绍了试桩检测方法及试桩、锚桩、工程桩的合理利用，并对其检测结果作了研究，指出该检测方法在施工或节约投资方面，均有广阔的前景。     

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.     

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。     

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.     

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.     

Removal of viable and inactivated Cryptosporidium by dual- and tri-media filtration

The limited efficacy of disinfectants, other than ultraviolet irradiation and ozonation, as a barrier against Cryptosporidium parvum in drinking water treatment has underscored the increased importance of oocyst removal by filtration. Currently, no reliable surrogates have been identified for C. parvum removal by filtration. As a result, evaluations of the Cryptosporidium removal by treatment operations have been performed using oocysts. It has typically been assumed that chemically inactivated oocysts are suitable surrogates for viable oocysts. Measurements of electrophoretic mobility, however, have shown that chemical inactivation changes the surface charge of Cryptosporidium oocysts. The present bench-scale research indicated that formalin-inactivated oocysts are reliable surrogates for viable oocysts during both stable filter operation and periods where filtration processes are challenged, such as coagulation failure. This finding is important because of the practical difficulties associated with using viable oocysts in filtration investigations. Poor coagulation conditions severely compromised removal of viable and inactivated oocysts by dual- and tri-media filters compared to stable operating conditions and filter ripening, emphasizing the importance of optimized chemical pre-treatment (coagulation) for the successful removal of oocysts during filtration. The treatment optimization experiments also indicated that tri-media filters offered only marginally higher oocyst removals than dual-media filters.     

Removal of Giardia and Cryptosporidium in drinking water treatment: a pilot-scale study

Giardia and Cryptosporidium have emerged as waterborne pathogens of concern for public health. The aim of this study is to examine both parasites in the water samples taken from three pilot-scale plant processes located in southern Taiwan, to upgrade the current facilities. Three processes include: conventional process without prechlorination (Process 1), conventional process plus ozonation and pellet softening (Process 2), and integrated membrane process (MF plus NF) followed conventional process (Process 3). The detection methods of both parasites are modified from USEPA Methods 1622 and 1623. Results indicated that coagulation, sedimentation and filtration removed the most percentage of both protozoan parasites. The pre-ozonation step can destruct both parasites, especially for Giardia cysts. The microfiltration systems can intercept Giardia cysts and Cryptosporidium oocysts completely. A significant correlation between water turbidity and Cryptosporidium oocysts was found in this study. The similar results were also found between three kinds of particles (phi=3-5,5-8 and 8-10 microm) and Cryptosporidium oocysts.     

Influence of land use and watershed characteristics on protozoa contamination in a potential drinking water resources reservoir

Relative changes in the microbial quality of Lake Texoma, on the border of Texas and Oklahoma, were investigated by monitoring protozoan pathogens, fecal indicators, and factors influencing the intensity of the microbiological contamination of surface water reservoirs. The watershed serves rural agricultural communities active in cattle ranching, recreation, and is a potential drinking water source. A total of 193 surface water samples were tested over a 27-month period to determine levels of parasite contamination. The overall occurrence of Cryptosporidium oocysts was higher in both frequency and concentration than Giardia cysts. Cryptosporidium oocysts were found in 99% and Giardia cysts in 87% of the samples. Although Cryptosporidium and Giardia occurrence were significantly but not strongly correlated, all other correlation coefficients including turbidity and total dissolved solids were non-significant. Statistically supportable seasonal variations were found suggesting that Cryptosporidium and Giardia were higher in summer and fall than in other seasons of the year. While Cryptosporidium levels were correlated with rainfall, this was not the case with Giardia. The maximum numbers for both protozoan parasites were detected from a site impacted by cattle ranching during calving season. Restriction fragment length polymorphism analysis was used for confirmation of Cryptosporidium in surface waters influenced by agricultural discharges. As we had expected, oocysts were of the bovine type indicating that the Cryptosporidium parvum detected in surface waters perhaps came from cattle living in the watershed.     

Occurrence of Cryptosporidium in agricultural surface waters during an annual farming cycle in lowland UK

A 17-month survey based on weekly testing for Cryptosporidium oocysts in surface waters draining a livestock farm on a Warwickshire (UK) estate has shown that the parasite is present throughout the year, with the highest frequency of occurrence and maximum concentrations during the autumn and winter. The 190 ha farm is managed as an exemplar for a teaching institution. There were up to 800 livestock present at peak times of year in the catchment of the stream draining the estate. Oocysts were concentrated from grab samples by a flocculation procedure, stained with monoclonal antibody and detected by fluorescence microscopy. Overall, 274/418 (66%) of samples were positive for Cryptosporidium. Where the stream passed from the estate, the occurrence was 79%, with mean and median oocysts/l of 0.48 and 0.2, respectively. Highest oocyst levels coincided with calving and increased wild animal numbers following breeding. There was no correlation of oocyst levels with rainfall or slurry spreading. Cryptosporidium was also frequently found in a pond on arable land (no livestock) indicating that wild animals alone contributed oocysts to surface waters. These results from a well managed livestock farm may represent a typical natural baseline for levels of occurrence and concentration of Cryptosporidium oocysts in surface waters of the lowland agricultural environment of the UK.     

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.     

Multi-scale Cryptosporidium/sand interactions in water treatment

Owing to its widespread occurrence in drinking water supplies and its significant resistance to environmental stresses, Cryptosporidium parvum is regarded as one of the most important waterborne microbial parasites. Accordingly, a substantial research effort has been aimed at elucidating the physical, chemical and biological factors controlling the transport and removal of Cryptosporidium oocysts in natural subsurface environments and drinking water treatment facilities. In this review, a multi-scale approach is taken to discuss the current state-of-knowledge on Cryptosporidium-sand interactions at a nano-scale, bench-scale and field-scale relevant to water treatment operations. Studies conducted at the nano-scale and bench-scale illustrate how techniques based on the principles of colloid and surface chemistry are providing new insights about oocyst-sand interactions during transport of Cryptosporidium oocysts in granular porous media. Specifically, atomic force microscopy and impinging jet experiments reveal the importance of oocyst surface biomolecules in controlling Cryptosporidium/sand interactions by a mechanism of steric hindrance. Traditional bench-scale column transport studies conducted over a broad range of experimental conditions highlight the role of physicochemical filtration and physical straining in the removal of oocysts from the pore fluid. Such experiments have also been used to evaluate the influence of biofilms formed on grain surfaces and the presence of natural organic matter on oocyst-sand interactions. Whilst filtration studies conducted at the plant-scale have been useful for evaluating the effectiveness of various materials as surrogates for Cryptosporidium oocysts, at this macro-scale, little could be learnt about the fundamental mechanisms controlling oocyst-sand interactions. This review of the literature on Cryptosporidium-sand interactions at different length scales points to the importance of combining studies at the plant-scale with well-controlled investigations conducted at the nano- and bench-scales. Furthermore, because oocyst surface properties play an important role in controlling the extent of interaction with sand surfaces, a thorough discussion of Cryptosporidium oocyst characteristics and electrical properties is presented.