PCR扩增石蜡包埋小鼠组织p53基因突变热点片段

目的 扩增石蜡包埋小鼠组织p53基因第5、7外显子。方法 用3种方法提取DNA，比较提取效果，用聚合酶链式反应（PCR）扩增相应片段。结果 用二甲苯脱蜡，蛋白酶K消化120小时可获得较好的DNA提取物。结论 用该提取物为模板扩增小鼠p53基因突变热点片段成功率高。     

垃圾渗沥液处理模式综合评价体系的建立

通过大量的资料搜集、文献检索和试验数据,系统地分析和总结影响垃圾渗沥液相关的因素,依据一定的评价原则,筛选出垃圾渗沥液处理系统评价指标,利用层次分析法理论构建一套初步可行的垃圾渗沥液评价指标体系;通过对专家咨询、实践经验以及对数据分析得到各指标的相对重要性,从而计算出各评价指标权重;在此基础上确定垃圾渗沥液评价标准,选择模糊数学法作为评价方法,构建渗沥液处理模式综合评价体系。     

从德国小蠊干标本中提取基因组DNA可行性的研究

目的拟建立用现场采集到的以及饲养过程中德国小蠊死亡后,晾干样本提取基因组DNA的方法。方法从现场环境中采集死亡的、体形较为完整的德国小蠊,晾干后,采用6种预处理方法后进行后续提取,并对所得基因组DNA质量进行吸光度、电泳与PCR扩增检测。结果从现场环境中采集到的德国小蠊干标本中获得了较高质量的基因组DNA,OD_(260/280)值可在1. 8～2. 0之间,1. 5%琼脂糖凝胶电泳在15 000 bp处有明显条带。以提取到的基因组DNA为模板可扩增出438 bp的β-actin基因和超过2 000 bp的钠离子通道基因VSSC片段。结论以本研究现场环境采集的体形较为完整的德国小蠊干标本为材料可以提取到基因组DNA,能满足至少2 000 bp的基因片段的PCR扩增要求,该方法在一定条件下有较强的可行性。     

PCR技术检测肉骨粉中牛源性成分的方法

建立了从肉骨粉中提取DNA的稳定、简便易操作的方法。根据牛线粒体DNA特异性片段设计引物 ,建立了PCR技术检测肉骨粉中牛源性成分的方法。同时 ,根据 1 8Sr RNA基因序列 ,设计通用引物来扩增所有真核生物的同源性基因片段 ,以此作为内源参照基因来检查从肉骨粉中提取模板的质量 ,避免假阴性结果。PCR反应循环参数是 96℃ 2min ;94℃ 40s,60℃ 50s,72℃ 60s ,35次循环 ;之后 72℃延伸 5min。肉骨粉中牛源性成分经PCR扩增出 2 71bp特异性基因片段     

可视化核酸等温扩增方法快速检测羊肉源性成分

目的 建立羊肉源性成分核酸等温扩增可视化快速检测方法.方法 用加热煮沸方法直接提取羊肉标本中的DNA;根据羊属特异性的Cytb基因序列的片段设计扩增引物,优化反应体系和反应条件,确立最佳等温扩增温度和扩增时间,对羊肉制品进行可视化等温扩增.并将建立的核酸等温扩增方法与荧光定量PCR法进行实际样品的检测.结果 建立的可视...     

乳汁提取基因组DNA及基因多态性检测研究

目的:探讨从乳汁中提取基因组DNA并进行相关基因多态性检测的新方法。方法:从乳汁中提取基因组DNA,并应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测△-6去饱和酶基因单核苷酸多态性。结果:提取的DNA纯度为1.8～1.9,DNA浓度为20～30 ng/μL。凝胶电泳显示,DNA条带完整,无拖尾现象,PCR扩增及酶切电泳图谱清晰。目的条带371 bp,经MSPI酶切后出现132 bp和239 bp两条片段。在人群中有3种基因型:纯合切开C/C,杂合基因C/T,纯合未切开T/T。结论:乳汁中可以提取完整的基因组DNA,并可以用于PCR扩增及限制性酶切。     

一种从蠕虫组织切片中提取DNA作PCR鉴定虫种的简便方法

目的建立一种实用于临床病理鉴别诊断组织切片内蠕虫虫种的分子病理学方法。方法制作已知的和从临床收集的多种蠕虫和宿主组织病理切片标本,用改良蛋白K消化法对各种蠕虫切片的白片、HE染色片和免疫组化片组织提取DNA;设计合成检测并殖吸虫、曼氏裂头蚴和猪囊虫各自特异性基因片段的引物,分别对各DNA提取物作PCR扩增,经凝胶电泳观察目的基因片段;比较分析三种蠕虫各自最小组织量检测的敏感性和特异性。结果对三种蠕虫所选用的引物均可扩增出清晰的特异性目的条带。用改良蛋白K消化法提取蠕虫DNA可PCR扩增出目的片段的敏感性均优于商品化试剂盒提取法,可提出DNA的最小组织面及厚度为:肺吸虫的为1.50 mm2,10μm;曼氏裂头蚴的为4.87 mm2,5μm;猪囊虫的为5.80 mm2,5μm。经10个临床标本检测均获得理想结果。结论本研究成功地探索出一种对病理切片中微量组织提取DNA作PCR鉴定虫种的简便方法,为病理诊断提供了进一步鉴别此3种蠕虫的分子病理学手段。     

金黄色葡萄球菌基因组DNA提取方法的比较研究

目的:对金黄色葡萄球菌DNA不同提取方法进行评价.方法:采用4种不同金黄色葡萄球菌DNA提取方法,比较提取得率和质量,对提取的DNA进行PCR扩增并评价效果.结果:在DNA提取之前加入70%乙醇可有效处理细菌细胞,增加细胞对裂解液的敏感性,再用溶菌酶处理能很好地被消化,获得了一种快速、简洁和有效的提取方法.结论:这是一种适用于金黄色葡萄球菌和其它病原细菌的有效提取方法(方法4),可用于金黄色葡萄球菌和其它病原细菌的分子生物学操作和快速诊断技术.     

兰州市首起甲型H1N1流感暴发的核酸检测

目的:鉴别一起不明原因导致的突发性发热事件病原体。方法:提取样本中的RNA后,使用针对于甲型H1N1流感病毒相关的不同浓度引物、探针和DNA聚合酶缓冲液等作为反应成份,按照不同的扩增程序,利用逆转录PCR(RT-PCR)和实时荧光定量PCR(real-time PCR)方法分别进行特异性DNA片段的扩增,对扩增产物的电泳图片和扩增图谱进行分析。结果:经RT-PCR方法检测后,分别扩增出235、527、153和80 bp的特异性片段,实时荧光PCR扩增曲线中,出现了与阳性对照一致的扩增图谱。结论:此次事件的病原体为甲型H1N1流感病毒,定性本次事件为甲型H1N1流感暴发事件。     

脱腐预处理对土壤总DNA提取质量的影响

目的探索一种提高土壤总DNA提取质量的简易脱腐方法。方法于2014年3月,采集河南省某市农田4个采样区的混合土样(07、08、1A、2A)。分别用脱腐预处理后试剂盒提取(预处理组)和直接试剂盒提取法(未预处理组)提取土样总DNA,对保守序列16S r DNA和非保守序列(抗生素抗性基因sulⅡ、tet M、tet C和1类整合子酶基因int I1)进行PCR扩增。采用Gene Genius凝胶成像系统定量分析软件Gene Tools进行灰度值分析。结果预处理组和未预处理组提取的各个土壤总DNA均可扩增出16S r DNA,且PCR产物灰度值差异无统计学意义(均P0.05);非保守序列sulⅡ、tet C和int I1基因的PCR扩增产物灰度值在两组中差异无统计学意义(均P0.05);预处理组tet M基因的表达水平高于未预处理组,差异有统计学意义(P0.05)。未预处理组的sulⅡ、tet M、tet C和int I1基因PCR扩增产物中均检测出了非特异性条带,而预处理组目的条带单一、较少非特异性条带。结论脱腐预处理方法提取的土壤总DNA更有利于后续分子生物学实验。     

电导法测定垃圾渗沥液中全盐量的探讨

为使垃圾渗沥液全盐量的测定方法快速、简便,以国家标准方法——重量法为参照,对10组渗沥液水样的电导率进行测定,建立全盐量和电导率的回归方程,并进行相关性分析。结果表明：2种测定方法建立的渗沥液全盐量回归方程为y=0.58x-0.42,相关系数r=0.999 7,标准差SD=0.047,线性关系较好。在一定条件下,可通过回归方程直接将电导率测试值转换为垃圾渗沥液全盐量,实现速测。但由于渗沥液的产生源、季节变化等原因,应对该方程进行相应的修正。     

城市垃圾填埋场渗滤液循环回灌处理技术

城市垃圾填埋场渗滤液经循环回灌处理后,污染物浓度大大降低,填埋场填埋垃圾的湿度增加,稳定速度加快。适量的表面回灌可有效减少渗滤液水量,降低处理规范。与传统渗滤液处理方法相比,此技术投资省,效果好,操作、管理简单,很有应用价值。     

浅析垃圾卫生填埋场渗滤液的处理

以汉沽区垃圾处理场可行性研究中的垃圾渗滤液处理方法为例,介绍了垃圾渗滤液的产生原因、流量的计算及处理方案。     

Assessment of sanitary landfill leachate characterizations and its impacts on groundwater at Alexandria

The total amount of solid waste generated in Alexandria is 2820 tons/d which increases to 3425 tons/day during summer. In the past, 77% of the collected solid wastes was open dumped. The open dumping sites did not have the minimum requirements for pollution control. Following the exacerbation of the problem, the Alexandria Governorate contracted a company to carry out the solid waste management. The contracted company transferred 75% of the daily generated solid wastes to a new constructed sanitary lanfill. The site receives a daily average of 1910 tons. The landfilling is performed by trench method in the form of cells. The produced leachate is discharged into two lined aerated lagoons. The biogas formed from biodegradation of landfilled solid wastes is burned and the produced heat is used for drying the lagoons leachate. The remaining residues are relandfilled. The study aims at assessment of the solid waste sanitary landfill leachate characterization and its impacts on the groundwater. The analysis of the collected data confirms that leachates from the landfill are severely contaminated with organics, salts, and heavy metals. The fluctuations in concentration levels of the different parameters were attributed to aging and thickness of waste layers, stage of decomposition, and re-landfilling of the concentrated residues from the drying lagoons. The concentrations of NH4-N (600 mg/l) indicated that the process of stabilization was still in the initial stages and attributed to the compaction process. The high BOD5 results (28,833 mg/l) indicated that the process of stabilization was in the initial stages which were very slow. The high COD results (45,240 mg/l) can be attributed to the compaction of the wastes which also retards the degradation of the solid wastes. The BOD and COD values indicated clearly severe contamination. The BOD5/COD ratio measured in the current study (0.64) indicated that the leachate of the present study was biodegradable and unstabilized, and required time and favourable conditions for anaerobic biodegradation. Heavy metals were lower compared with what have been observed in other countries. Re-landfilling of the residue after drying the leachate in lagoons and the short time of biodegradation in the landfill site were factors which effected the high strength of most of the parameters concentrations of the leachate. Assessment of groundwater contamination through piezometer wells around the active cells indicated that there was no contamination from the leachate to the groundwater surrounding the site. The study recommended emphasizing the importance of adjusting the biodegradation factors, the monitoring program, the prohibition of disposing heavy metals, determination of the leachate generation rate, and treatment of leachate.     

用SBR法处理寒冷地区垃圾场渗滤液

牡丹江市郭家沟垃圾场，垃圾渗滤液通过贮存池、SBR（Sequence Batch Reactor，序批式活性污泥法）反应池、混凝沉淀池等处理工艺，使渗滤液达到国家排放标准，说明SBR法适用于寒冷地区垃圾填埋场的渗滤液处理。     

An effective method for extracting DNA from contaminated soil due to industrial waste

Recently, labor accidents caused by toxic gas production such as H2S and CH4 have happened at some waste disposal sites and illegal dumping sites in Japan. The gas production has been known to be end products of bacterial respiration. In order to investigate a microbial flora in industrial waste contaminated soil by a DNA-based detection method, we developed a simple DNA extraction method. The method is based on lysis with sodium dodecyl sulfate (SDS) and a high concentration (4.0 mg/ml) of lysozyme at room temperature. DNA yields increased according to the concentration of lysozyme (0-2 mg/ml), although the cell lysis occurred at comparable efficiency independent of the lysozyme concentration. Interestingly, bovine serum albumin and insulin (4 mg/ml) also contributed high DNA yields. This suggests proteins play an important role at high concentration on a DNA extraction process, presumably by suppressing DNA degradation or adsorption. DNA yields from three contaminated soil samples at 0.3, 1.5 and 3 m in depth by using our method were significantly higher than those extracted by using a widely-used conventional method which did not contain lysozyme. A novel and simple DNA extraction method using high concentration of protein was established for a DNA-based detection method of microbial flora in soil.     

SBR法处理垃圾渗滤波试验研究

利用ＳＢＲ法对城市生活垃圾卫生填埋场垃圾渗滤液进行处理,渗滤液在ＳＢＲ法处理前进行吹脱处理,以污染物主要指标ＣＯＤ、氨氮作为处理程度指示。经过试验处理后,渗滤液ＣＯＤ值大大降低,其去除率可达 ９０％以上,氨氮的出水值低于国家废水综合排放二级标准。     

垃圾填埋场的结构设计及渗漏分析

介绍了国内外垃圾填埋场防渗技术的研究现状及存在问题。借鉴发达国家的应用经验和研究成果,对我国垃圾填埋场的结构设计进行分析,包括填埋场防渗衬垫的选材,结构布置和施工方法,填埋场封顶系统的设计,渗沥液收集与排放系统和填埋气收集与处理系统的设计。预估填埋场渗沥液渗漏量的计算方法,分析了渗沥液在压实黏土、土工膜中的渗漏特点,提出了对各种防渗衬垫的优化布置方法。     

Field survey of enteric viruses in solid waste landfill leachates.

Because municipal solid waste may contain fecal material from a variety of sources, there is concern that the leachate discharged from some solid waste landfills may contain enteric pathogens, including enteric viruses. In this study, 22 leachate samples from 21 different landfills in the United States and Canada were examined for enteric viruses. The sites represented a broad range of conditions for solid waste landfills and the leachate samples ranged from 10.3 to 18 liters in volume. Enteric viruses were found in only one of the 22 leachate samples examined. Two viruses, identified as poliovirus types 1 and 3, were found in an 11.8 liter sample obtained from a site where solid waste landfill practice was deficient. The low levels of enteric viruses detected in field samples of raw leachate and the opportunities for further reductions in the virus concentration of leachates by such processes as thermal inactivation, removal by soil and dilution in ground and surface waters, suggest that leachates from properly operated solid waste landfills do not constitute an environmental or public health hazard due to enteric viruses.     

Organic contaminants in the Jakusevec landfill and their impact on groundwater quality

Landfilling is probably, the most popular disposal method for the management of domestic and industrial waste. Unfortunately, many landfills around the world do not include leachate collection systems and present a considerable risk to the underlying aquifers. There have been numerous reports on groundwater contamination in the vicinity of unprotected landfills, paying particular attention to specific organic contaminants. This problem is especially pronounced in landfills which, in addition to ordinary domestic refuse, contain waste of industrial origin. Jakusevec, the main landfill of the city of Zagreb, belongs to this category. The recently completed remediation programme for Jakusevec included comprehensive investigations with the aim to quantify various organic contaminants in solid waste and establish their possible infiltration into the underlying soils and groundwater. A detailed analyses using chromatographic and mass spectrometric techniques made it possible to identify a number of contaminants. According to their origin, the identified compound classes can be divided into the two main categories: markers of biological waste and its microbial transformation and markers of anthropogenic waste. Our investigations have shown that the composition of organic contaminants changes dramatically during the vertical transport from the solid waste through the unsaturated zone of soil to aquifer sediments, which involves both physicochemical and biotransformation processes. The vertical transport of polar organic contaminants was very efficient and led to a significant contamination of the aquifer in the vicinity of the landfill.