应用随意引物PCR技术克隆猪带绦虫特异的基因探针

筛选和克隆猪带绦虫基因组特异的ＤＮＡ片段作为探针，建立脑囊虫病的基因诊断方法。以一１９ｎｔ的寡核苷酸为探针，通过随意引物ＰＣＲ反应扩增猪带绦虫基因组ＤＮＡ，分离出数个不同大小的扩增片段。分别以其中０．７，０．８ｋｂ片段为探针作ＤＮＡ斑点杂交分析，证实此２片段为猪带绦虫基因组所共有，在人和猪基因组中未发现有同源序列存在。猪带绦虫基因组特异ＤＮＡ探针的分离和克隆将使猪带绦虫所致寄生虫病的基因诊断成为可能。     

淡色库蚊和缺乏库蚊基因组多态性DNA的研究

本文应用随机引物扩增多态性ＤＮＡ聚合酶链反应技术（ＲＡＰＤ－ＰＣＲ）筛选出３种引物，对淡色库蚊，致乏库蚊的细胞基因组ＤＮＡ进行扩增，分别扩增出不同条带状数和分子大小的多态ＤＮＡ图谱，两种库蚊细胞的基因组ＤＮＡ扩增产物除了有相同分子大小的ＤＮＡ带外，还有一半条带为各自独特的条带，用淡色库蚊成蚊作ＲＡＰＤ－ＰＣＲ的扩增带与相应的细胞株产生的主要条带相符，实验结果表明，此技术方法简便，快速，精确，可用于     

嗜肺军团菌聚合酶链反应检测方法及其应用研究

根据嗜肺军团菌基因组ＤＮＡ的种特异性ＤＮＡ片段序列，合成一对引物进行聚合酶链反应（ＰＣＲ）。经琼脂糖凝胶电泳、ＥＢ染色结果表明，一条８７０ｂｐ的核苷酸区带为嗜肺军团菌１～１４血清型菌株所共有。ＰＣＲ检测水中军团菌，其敏感性为３５０ｃｆｕ／ｍｌ，而用同位素标记探针、斑点杂交法检测，其敏感性为４３ｃｆｕ／ｍｌ。ＰＣＲ检测人工感染嗜肺军团菌的豚鼠组织标本，阳性率为８３．３％，而细菌分离培养的阳性率仅为２６．６％。在现场调查中，用ＰＣＲ法初步验证了一起由Ｌｐ１０引起的军团菌病爆发。上述结果表明，ＰＣＲ法能快速、敏感、特异性检测嗜肺军团菌感染。     

吸烟与肺组织DNA加合物形成关系的研究

目的探讨吸烟与人肺组织ＤＮＡ加合物含量的关系以及在肺癌患者与非肺癌患者之间、男女性之间有无差别。方法提取同期住院原发肺癌患者与非肺癌患者（男各１９例，女各１８例）手术切除正常肺组织ＤＮＡ，采用３２Ｐ后标记方法测定ＤＮＡ加合物含量。结果吸烟可明显地引起肺组织ＤＮＡ加合物含量增高。如果控制吸烟因素，男女之间，非肺癌患者与肺癌患者之间的肺组织ＤＮＡ加合物水平差异无统计学意义。结论人体中ＤＮＡ加合物水平与生物学效应之间的关系尚需积累更多的资料加以阐明     

性别、年龄因素对人肺组织DNA加合物形成量影响的探讨

探讨了ＤＮＡ加合物的量在人肺组织中随年龄、性别的分布。实验结果表明：年龄与肺组织中ＤＮＡ加合物的相关系数很低，不同年龄组ＤＮＡ加合物的平均相对加合物标记（ＲＡＬ）值无显著性差异。男性肺组织中ＤＮＡ加合物的量显著高于女性（Ｐ＜０．０５），除与吸烟因素有关外，男女肺组织中ＤＮＡ加合物量的差异也不能排除内分泌因素的影响。     

非逆转录RNA病毒的DNA形式

逆转录病毒由于编码逆转录酶,在感染的宿主细胞中很容易检测到病毒ＤＮＡ,同时病毒ＤＮＡ也能整合到宿主基因组中,但令人惊奇的是在淋巴细胞性脉络丛脑膜炎病毒（ＬＣＭＶ）,丙型肝炎病毒（ＨＣＶ）和丁型肝炎病毒（ＨＤＶ）等非逆转录ＲＮＡ病毒感染的宿主细胞中也能检测到病毒ＤＮＡ,这种ＤＮＡ形式甚至在还存在于宿主细胞的基因组中。本文这方面的有关研究情况作了综述。     

新假说:创伤病人新的预后标志:血浆DNA测定

新近研究表明血浆ＤＮＡ可作为诊断与监测工具 ,本文提出假说 :创伤病人血浆ＤＮＡ水平增高可作为此种病人预后的指标。对 84名钝器伤病人以实时定量ＤＮＡ分析法测定β -球蛋白基因。结果对照组 ,轻度 /中度创伤 (损伤严重性评分 <16;ｎ =4 7)和重度创伤 (损伤严重性评分≥ 16;ｎ =37)组 ,血浆ＤＮＡ平均浓度分别为 3154千基因组当量 (暂译名 ,Ｋｉｌｏ ｇｅｎｏｍｅ -ｅｑｕｉｖａｌｅｎｔｓ) /Ｌ ,13818千基因组当量 /Ｌ和18130 3千基因组当量 /Ｌ。血浆ＤＮＡ浓度与合并急性肺损伤 ,急性呼吸窘迫征或创伤致死有关 ,它可比无此种合并症者…     

环氧乙烷对大鼠DNA损伤机理的研究

为探讨环氧乙烷（ＥｔＯ）对ＤＮＡ分子水平的损伤作用，采用血红蛋白基因特异性的单引物，对用ＥｔＯ染毒不同时间的Ｗｉｓｔａｒ雄鼠和对照个体的肝、肾、睾丸等组织的ＤＮＡ进行半随机ＰＣＲ扩增。结果显示：在吸入染毒（８００ｍｇ／ｍ３）６～９周条件下，ＥｔＯ可引起肝、肾、睾丸等组织不同程度的ＤＮＡ损伤，其中睾丸组织ＤＮＡ损伤最为严重。     

乙、丙型肝炎病毒重叠感染的慢性肝病患者肝组织和血清中的病毒检测

为了探讨乙型和丙型肝炎病毒（ＨＢＶ和ＨＣＶ）重叠感染者病毒相互干扰现象，对４４例患者血清检测诊断为ＨＢＶ和ＨＣＶ感染的慢性肝病患者，用原位杂交法检测了肝组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。２８例患者肝组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ同时阳性，两者阳性信号分布类型比较无明显差异（Ｐ＞０．０５），血清ＨＢｅＡｇ阳性和阴性组中肝组织中的ＨＢＶＤＮＡ和ＨＣＶＲＮＡ检出率无明显差异（Ｐ＞０．０５）。８例血清ＨＢｓＡｇ阴性者，肝组织ＨＢＶＤＮＡ阳性５例，ＨＣＶＲＮＡ阳性３例。结果提示，ＨＢＶ和ＨＣＶ重叠感染肝组织损伤加重，可能诱导一种病毒呈现潜伏状态，但不是病毒清除。     

新型生物组织透明剂代替二甲苯在病理制片技术中的应用研究

目的探讨BT生物组织透明剂代替二甲苯进行组织石蜡制片的效果。方法应用BT生物组织透明剂代替二甲苯作为透明剂用于组织透明制作组织蜡块,并用于石蜡切片染色前的脱蜡和染色后的切片透明。结果用本法制作的组织蜡块,经切片脱蜡后进行的常规HE、组织化学和免疫组化染色,组织细胞形态完好,无明显收缩;组织中的各种成分和抗原保存完好,染色效果好,与使用二甲苯制作的切片质量无明显差别。结论BT生物组织透明剂能够代替二甲苯在组织脱水处理过程中对组织进行透明,也可用于常规HE、组织化学和免疫组化染色前的石蜡切片脱蜡和染色后的切片透明,并符合实验室环保的要求。     

长春龙嘉国际机场口岸蜚蠊本底监测分析及携带病原菌基因组DNA提取方法初探

目的掌握长春龙嘉国际机场口岸蜚蠊本底数据,对蜚蠊携带病原菌基因组DNA提取方法进行比较。方法在2008年4至2009年3月期间,采用盒式诱捕法捕获蜚蠊,分别采用煮沸法、试剂盒法、SDS法三种方法提取蜚蠊表面携带病原菌的基因组DNA,并进行统计分析。结果共捕获蜚蠊3957只,全部为德国小蠊,口岸蜚蠊密度为1.36只/盒,其中餐饮区蜚蠊密度最高为4.08只/盒;1月份和7月份为蜚蠊密度高峰期。三种方法提取病原菌基因组DNA的纯度和浓度具有显著性差异。结论应加强在长春龙嘉国际机场口岸蜚蠊的防控工作,防止口岸蜚蠊密度的上升;SDS方法可作为蜚蠊携带病原菌基因组DNA的提取方法。     

An improved method for efficient isolation and purification of genomic DNA from filamentous cyanobacteria belonging to genera Anabaena, Nodularia and Nostoc

The aim of this work was to develop an improved method for isolation and purification of genomic DNA from filamentous cyanobacteria. The method described here employs a modified phenol extraction-based procedure. It allowed us to obtain a high yield (60–620 μg/g wet weight, depending on the cyanobacterial strain) of pure and undegraded genomic DNA (A260/A280 ratio of about 1.8 and A260/A230 ratio of about 2.0). Genomic DNA, isolated from cyanobacteria belonging to the genera Anabaena, Nodularia and Nostoc has been successfully used for construction of gene libraries. Thus, this method can be used in procedures requiring highly purified cyanobacterial DNA.     

随机扩增多态性DNA在淋球菌基因分型中的应用

目的 建立随机扩增多态性DNA(RAPD)对淋球菌进行基因分型的方法。方法 用4种不同的预处理方法提取淋球菌基因组DNA,对其优劣进行比较;并运用RAPD对淋球菌菌株进行区分及对经性伴传播的淋病病例进行RAPD指纹图谱比较。结果 运用经典的十六烷基三乙基溴化铵法可以抽提较完整的基因组DNA,获得良好的RAPD指纹图谱;各菌株的RAPD指纹图谱间有明显不同DNA多态性;性伴传播的病例中获得了非常相似的RAPD指纹。结论 选择最佳的DNA抽提技术,运用RAPD可以对淋球菌进行有效基因分型,并可用于分子流行病学对传染源的追踪。     

立氏立克次体实时荧光定量聚合酶链反应检测方法的建立

目的采用TaqMan-MGB探针建立立氏立克次体实时荧光定量聚合酶链反应（PCR）检测方法.方法依据立氏立克次体外膜蛋白B基因序列设计引物和探针,以克隆的ompB基因片段为DNA模板,在ABI 7900型荧光定量PCR检测仪上建立实时荧光定量PCR检测方法.结果建立的定量标准曲线Ct值与模板拷贝数呈线性关系（R2=0.996）,最低检测浓度为5拷贝/μl;用荧光定量PCR方法检测其他相关立克次体和常见非立克次体病原菌,检出结果均为阴性.用该方法检测立氏立克次体感染的豚鼠血液标本、小鼠脾脏标本及细胞培养标本,检测的结果与立氏立克次体感染相关.结论研究建立的检测立氏立克次体实时荧光定量PCR方法具有高特异性和高敏感性,适用于快速检测各种样本中的立氏立克次体和立氏立克次体感染早期的实验室诊断.     

通过高温变性快速提抽DNA的方法学研究

目的 探讨从血清中快速抽提基因组DNA片段的方法。方法 应用高温变性和离心法处理人血清标本。并使用各种引物进行扩增以检测抽提效果。结果 本方法抽提的DNA作为模板，可以稳定扩增出400个碱基对以下的特异性基因组DNA片段。结论 高温变性和离心法抽提人血清DNA。操作简便，成本低廉，抽提出的基因组DNA片段能够作为扩增一定大小目的DNA片段的模板。     

A DNA probe to distinguish the species Anopheles quadriannulatus from other species of the Anopheles gambiae complex

DNA probes used previously to distinguish the species Anopheles gambiae sensu stricto, An.arabiensis, An.melas and An.merus were tested against An.quadriannulatus. Using these DNA probes, An.gambiae s.s. and An.quadriannulatus were indistinguishable. A genomic library was constructed for An.quadriannulatus. Differential screening of this genomic library with An.gambiae s.s. and An.quadriannulatus genomic DNAs identified a species-specific, repeated DNA sequence. When used as a hybridization probe, this DNA sequence clearly distinguished An.gambiae s.s. from An.quadriannulatus. A simplified protocol for the use of DNA probes is described which may be used to identify material squashed directly on to nitrocellulose filter paper.     

Testing the retrovirus hypothesis of manic depression and schizophrenia with molecular genetic techniques.

Crow's viral hypothesis of schizophrenia proposes that psychosis may be the result of mutagenesis caused by viral integration or transposition in human genomic DNA. Molecular genetic techniques can be used to systematically investigate this hypothesis. In a study of genomic lymphocyte DNA unexpected DNA polymorphisms which were probably insertions and deletions were found in specific human genomic retroviral (proviral) related sequences. However these changes were found exclusively in normal Icelandic individuals and are probably of evolutionary origin. The extent to which human retroviral insertion and deletion has taken place and the mobility of such sequences will help in understanding their evolutionary origin and might provide a source of polymorphic marker sequences that could be used in genetic linkage studies of disease.     

人血凝块提取基因组DNA碘化钾法建立

目的 通过与酚/氯仿法比较,建立一种快速、经济、高效从人血凝块提取基因组DNA的简易方法.方法采用双蒸水低渗破碎红细胞,碘化钾直接、快速裂解白细胞及其核膜,氯仿/异戊醇沉淀蛋白质及残存细胞碎片,最后经异丙醇和乙醇沉淀基因组DNA.结果 采用该法提取的基因组DNA浓度为(46.4±8.8)mg/L,吸光度值A260/A280为(1.79±0.23),与酚/氯仿法(46.1±8.3,1.78±0.22)比较差异无统计学意义(P>0.05);2种方法 提取基因组DNA凝胶电泳条带完整,PCR扩增目的 条带完整,能够满足分子生物学实验要求.结论 碘化钾法是一种快速、简便、经济、高效提取人血凝块基因组DNA的方法,可以广泛运用于大规模人群基因组学研究.     

Folate supplementation increases genomic DNA methylation in the liver of elder rats

The availability of folate is implicated as a determinant of DNA methylation, a functionally important feature of DNA. Nevertheless, when this phenomenon has been examined in the rodent model, the effect has not always been observed. Several reasons have been postulated for the inconsistency between studies: the rodent is less dependent on folate as a methyl source than man; juvenile animals, which most studies use, are more resistant to folate depletion than old animals; methods to measure genomic DNA methylation might not be sensitive enough to detect differences. We therefore examined the relationship between folate and genomic DNA methylation in an elder rat model with a newly developed method that can measure genomic DNA methylation sensitively and precisely. Thirty-nine 1-year-old rats were divided into three groups and fed a diet containing 0, 4.5 or 18 mumol folate/kg (folate-deplete, -replete and -supplemented groups, respectively). Rats were killed at 8 and 20 weeks. At both time points, mean liver folate concentrations increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend <0.001) and by 20 weeks hepatic DNA methylation also increased incrementally between the folate-deplete, -replete and -supplemented rats (P for trend=0.025). At both time points folate-supplemented rats had significantly increased levels of DNA methylation compared with folate-deplete rats (P<0.05). There was a strong correlation between hepatic folate concentration and genomic DNA methylation in the liver (r 0.48, P=0.004). In the liver of this animal model, dietary folate over a wide range of intakes modulates genomic DNA methylation.     

A modified pulsed-field gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difficile polymerase chain reaction ribotype 001

A modified pulsed-field gel electrophoresis (PFGE) protocol was developed and applied to 50 isolates of the UK epidemic strain of Clostridium difficile, polymerase chain reaction (PCR) ribotype 001, to develop a PFGE-based subtyping scheme. This protocol overcame the inherent DNA degradation problems associated with typing this strain of C. difficile by this method, and whole genomic digestion with SmaI restriction enzyme yielded seven distinct and reproducible PFGE banding patterns. Modified PFGE is an appropriate method for subtyping C. difficile PCR ribotype 001 that could be used to improve epidemiological investigations.