儿童呼吸道腺病毒基因分型方法的建立及其应用

目的 利用PCR技术,建立引起儿童呼吸道感染的腺病毒的基因分型方法,便于推广和应用.方法 分析GenBank中不同型别腺病毒的六邻体基因序列特点,设计不同型腺病毒的特异性引物,以PCR方法进行基因分型,建立基因分型方法,并利用此方法对广州市2004年7月某幼儿园流行的腺病毒感染进行了基因分型.结果 建立了呼吸道腺病毒的基因分型方法.广州市2004年7月某幼儿园流行的腺病毒为3型腺病毒.结论 PCR方法可进行腺病毒的基因分型,且简便、结果可靠,便于推广应用.     

PCR－小卫星DNA指纹图在异基因造血干细胞移植植活指标监测中的应用

为了解异基因骨髓移植治疗某些遗传及恶性血液性疾病的植活监测指标，用聚合酶链式反应（ＰＣＲ）扩增具有高度多态性的小卫星ＤＮＡ３３．１、３３．６位点，联合地高辛标记的位点特异性寡核苷酸探针杂交的方法，以小卫星ＤＮＡ指纹图为特异性遗传标志，对６例异基因骨髓移植，１例脐血移植进行植活指标监测，６例获得供者源性细胞植活证据。结果显示：小卫星ＤＮＡ指纹图个体特异性强，可作为监测异基因造血干细胞移植植活的可靠格标，尤其是同血型，同性别，ＨＬＡ配型表型全相合的移植；该方法程序简单，操作方便，灵敏度高，可达０．５％，最早获得植入证据的时间是＋１５天，仅需模板ＤＮＡ１００ｎｇ；如果扩增位点增加，可提高个体识别能力。     

HLA—DRB1＊04等位基因亚型的DNA分型

采用顺序特异引物聚合酶链反应技术（ＰＣＲ－ＳＳＰ）检测我国汉族人群ＨＬＡ－ＤＲＢ１＊０４等位基因亚型频率，并与白种人群进行比较。利用５′端公用引物与３′端８个特异性引物进行ＰＣＲ反应，可准确分辨出１１个ＤＲＢ１＊０４等位基因，耗时４小时，无假阳性和假阴性结果。８５份随机样本的分型结果显示，我国汉族人群ＤＲＢ１＊０４基因亚型的频率与白种人之间存在非常显著性差异。     

Cat red nucleus activity preceding movement depends on initiation conditions

Summary The activity of 98 Red Nucleus neurons was recorded in 3 cats operantly conditioned to perform a ballistic forelimb flexion movement triggered after a brief sound in a simple Reaction Time condition, or Delayed after the same sound in the presence of a tone cue. Fifty-eight task related neurons presented changes of activity in either one or both conditions. Forty-four of them were studied quantitatively and classified in 3 categories: 1) only 16% of the units presented similar changes of firing preceding the triggered or delayed movement; 2) most units (55%) presented different changes of activity in the two conditions: in the Delayed condition, the activation occurred earlier before the movement, and/or the change in magnitude was reduced or the pattern of activity was modified; 3) moreover, for 29% of the units, the change of activity observed before movement in the Reaction Time condition was severely reduced or even absent in the Delayed condition. For some of these neurons a building-up of activity was observed very early in the Reaction Time condition, during the preparatory period, well before the occurrence of the conditioned stimulus. These results show that the Red Nucleus activity preceding a movement is clearly dependent on its initiation conditions. The distinct patterns of unit firing observed in the Reaction Time condition and in the Delayed condition are tentatively related to the different preparation and initiation constraints determined by the behavioral conditions.     

用PCR扩增tim基因检测蓝氏贾第鞭毛虫

采用聚合酶链反应 (PCR)对蓝氏贾第鞭毛虫 (Giardialamblia)的磷酸丙糖异构酶 (triosephosphateisomerase ,缩写为tim)基因进行特异性扩增 ,结果扩增出 1条 6 83bp的DNA片段。此方法的特异性可高达10 0 % ,而其它DNA样本 ,如日本血吸虫 (Schistosomajaponicum)、刚地弓形虫 (Toxoplasmagondii)、微小隐孢子虫 (Cryptosporidiumparvum)、溶组织内阿米巴 (Entamoebahistolytica)、旋毛虫 (Trichinellaspiralis)和阴道毛滴虫 (Trichomonasvaginalis) ,以及人体血细胞等均未出现扩增反应。本法的敏感性也很高 ,可检测到0 4pg贾第虫包囊的DNA。 13株来自不同地理位置和 或宿主的贾第虫DNA样本在PCR中均各产生 1条长为 6 83bp的目的片段。上述结果表明本实验建立的检测贾第虫的PCR方法有效     

用PCR技术产前诊断人巨细胞病毒感染

用ＥＬＩＳＡ法筛选早孕妇女血人巨细胞病毒ＩｇＭ（ＨＣＭＶ－ＩｇＭ），在孕１２￣２０周用聚合酶链式反应（ＰＣＲ）技术检测其羊水中ＨＣＭＶ－ＤＮＡ，对胎儿巨细胞病毒感染作出产前诊断。在３８４例孕妇中，筛选出血ＨＣＭＶ－ＩｇＭ阳性２６例为实验组，其羊水ＨＣＭＶ－ＤＮＡ检出率为３４．６２％；从２５８例ＨＣＭＶ－ＩｇＭ阴性中选出２０例为对照组，其羊水ＨＣＭＶ－ＤＮＡ检出率为１０．０％，两组比较有显著性差异（     

PCR 检测产 A、B、E、F 和 G 型肉毒神经毒素梭菌的神经毒素基因

肉毒神经毒素分为Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ和Ｇ七个血清型，其中Ａ、Ｂ、Ｅ和Ｆ型引起人类肉毒中毒。为此，选取肉毒神经毒素的保守序列设计了一对简并引物，对１８株肉毒梭菌和８株相关梭菌进行了检测，表明该引物可特异扩增Ａ、Ｂ、Ｅ、Ｆ和Ｇ型肉毒梭菌及产Ｅ型肉毒神经毒素的丁酸梭菌，产物为２６４ｂｐ，敏感性达１０ｐｇ细菌ＤＮＡ。采有酚－氯仿抽提法、固相载体捕获法和热裂解法处理产毒菌株，结果表明热裂解法安全、简便、快速，所制模板扩增效果明显。因此，此ＰＣＲ方法可用于快速敏感地检测引起人类肉毒中毒的梭菌。     

Detection of cytomegaloviral DNA in human milk cells and cell free milk whey by nested PCR

Human cytomegalovirus (HCMV) DNA can be detected in different compartments of human milk. A protocol for the preparation of milk whey free of fat and cells for the detection of human cytomegalovirus (HCMV) by nested PCR is presented. This is based upon the experience of the separation of more than 200 milk specimens of healthy seropositive breast feeding mothers. HCMV DNA could be detected in freshly centrifuged and filtrated milk whey specimens without contamination by cellular DNA. In limiting dilution experiments using HCMV plasmid DNA, the effect of different DNA extraction procedures from native milk and milk whey on the detection limit of cytomegaloviral DNA was demonstrated. About 200 viral genome equivalents/ml in milk whey or native milk were detectable by classical organic phenol/chloroform extraction or a spin column method, respectively. The detection of viral DNA in milk cells depended on a minimum number of milk cells (10⁵–2×10⁵) available for DNA extraction. In contrast to the findings of cytomegaloviral DNA in native sera or plasma of immunosuppressed patients we failed to amplify low level viral DNA from native breast milk by nested PCR due to an inhibition of Taq polymerase by lipid components. Finally, the course of cell associated and cell free DNAlactia was monitored. Analyzing sequential milk specimens, in some cases the presence of HCMV DNA in colostrum could be demonstrated. DNAlactia of milk cells and whey was partially discordant. Onset (week 1–4 after delivery) and duration (2 weeks up to more than 3 months) of DNAlactia showed distinct individual patterns. The methods described, allow further analysis of the mechanisms involved in the postnatal HCMV transmission by breast feeding seropositive mothers.     

结核性淋巴结炎的组织细胞反应性增生变型

应用结核杆菌ＤＮＡ１２３ｂｐ特异性序列片段为靶序列的多聚酶链反应（Ｍ·ＴＢ－ＰＣＲ）技术、ＢＣＧ免疫组化（ＢＣＧ－ＩＨＣ）技术和抗酸染色（ＡＦ）方法，对３８例呈现组织细胞反应性增生－碎屑样坏死－嗜中性白细胞渗出病变的淋巴结石蜡包埋组织进行了分支杆菌／结核杆菌的回顾性检测。三种方法的综合阳性率为５２．６％（２０／３８例）。ＡＦ、ＢＣＧ－ＩＨＣ和Ｍ·ＴＢ－ＰＣＲ的各自阳性率分别为０．８％、２６．３％和５０％。研究结果表明：（１）在按本文标准选择的淋巴结“组织细胞反应性增生”病变中，有半数病例与结核杆菌的感染有关，即结核性淋巴结炎可呈现“组织细胞反应性增生”之类的变型；（２）ＰＣＲ技术在结核性淋巴结炎的病原学诊断上具有重要价值。     

SEN病毒ORF2基因序列和抗原性的初步分析

目的对SEN病毒(SENV)开放阅读框(ORF)2基因进行序列测定分析和原核表达,并对表达蛋白进行抗原性分析。方法用聚合酶链反应(PCR)方法扩增SENVORF2基因,对该基因进行序列测定、系统进化分析,双酶切扩增产物与原核表达载体pRSETB构建成重组质粒,诱导表达后进行SDSPAGE和免疫印迹分析。结果该株SENVORF2与北京株(AY072045)核苷酸同源性为97%,氨基酸同源性为59%;与日本株(AB059352)同源性分别为90%和56%。含有SENVORF2质粒的菌株表达相对分子质量约为19×103的融合蛋白,免疫印迹证明该融合蛋白能与SENVDNA阳性血清发生特异反应。结论表达了158个氨基酸的SENVORF2原核表达蛋白具有一定的抗原活性。