恙虫病东方体在无生命培养基内生长的研究

目的　恙虫病东方体 (Orientiatsutsugamushi;Ot)被认为属专性细胞内寄生。特别是制备大量抗原培养难的问题 ,是近一个世纪以来人们渴望解决的难题。为证实是否能在无生命培养基生长。方法　用Vero -E6个单层组织细胞培养OtJL - 90株与标准Gilliam株 ,在合适时取该培养物接种于Ⅲ号无生命固体培养基培养 ,置 36℃ (± 1℃ )培养 18- 2 4h。结果　可见生长一群无色透明或半透明光滑、湿润、圆形或枫叶样小菌落 ,并产生一种难闻的臭味。两种方法培养物分别做了生物学性状鉴定、形态染色、小白鼠致病力、血清学特异性间接免疫荧光检测。各株Ot经两种培养获得培养物特性基本一致。结论　Ot能在细胞内生长也能在无生命培养基内生长 ,解决了Ot大量抗原培养难的困扰。     

4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.     

4种病原菌特异基因片段阳性蜱的16S rRNA基因克隆文库分析

Objective To develop the method of 16S rRNA gene clone library for tick bacterial flora analysis, and to analyze the detection effective of pathogens in tick and capacity of bacterial flora diversity. Methods Primers were designed according to the specific gene of Borrelia burgdorferi, Bartonella henselae, Anaplasma phagocytophilum, Ehrlichia chaffeensis and templates were choosen by positive PCR result to amplify the DNA extracted from the ticks. One set of primers targeting 16S rRNA gene conserved region were chosen to amplify certain fragments, DNA extraction, PCR reaction, cloning and sequencing. Nucleotide sequences were compared with GenBank database. Calculated Coverage values of clone library and Shannon-Wiener diversity index. Results Sixteen defined genus-or species-bacteria were detected in 103 valid sequences. Eight species were edge type (Clone No. > 5). Three kinds of pathogens were identified (Borrelia burgdorferi, Bartonella henselae and Rickettsia sp). Three kinds of pathogens were not edge type(Clone No. < 5). Coverage value was 96.11%, and Shannon-Wiener index was 2.40. Analysis results of cloning sequence showed that tick-parasitic bacteria mainly were α and γ deformation mycetes which accounted for 56.25% (9/16). Conclusions The 16S rRNA gene sequences technology could make relative quantitative of bacterial flora, and detect many kinds of pathogens in tick. It's a good method for detection of pathogens and bacterial flora analysis.     

基因克隆技术在普氏立克次体研究中的应用

我们用鸟枪法，将普氏立克次体ＤＮＡ经酶切，连接，用ｐＵＣ１９为载体，大肠杆菌ＪＭ１０３为受体菌，将酶切后的连接产物转化到大肠杆菌ＪＭ１０３中去，用酶联法、改良补体结合法筛选克隆子。这样得到２株酶联效价为１∶１２８０的阳性克隆子，它们与普氏立克次体免疫血清呈阳性反应，具有种特异性     

22例老年甲状腺功能亢进症临床特点分析

目的　探讨老年甲状腺功能亢进症的临床特点。方法　对 2 2例老年甲亢与 2 6例青年甲亢的临床资料进行对比和分析。结果　老年组食欲亢进低于青年组 (P <0 .0 1) ,烦躁及震颤较青年少见 (P <0 .0 5 ) ,怕热多汗、消瘦乏力及腹泻两组相比无显著性差异 ,老年组窦性心动过速、早搏或房颤较青年组多见 (P <0 .0 5 ) ,老年组甲状腺明显低于青年组 (P <0 .0 0 1) ,突眼两组无显著性差异。结论　老年甲亢症状不典型 ,易漏诊及误诊 ,值得重视。     

云南玉溪地区首次发现斑点热

玉溪市红塔区地处云贵高原西南、云南省中部,属中亚热带半湿润冷冬高原季风气候,辖2乡7镇30多万人口。1999年6～8月对7个镇不明原因发热病人(病例组)进行调查,以非发热病人作为对照组,采用微量室温补体结合法,用西伯利亚立克次体抗原检测血清特异性ＩｇＧ抗体,抗体效价≥1∶8为阳性;根据抗体滴度、排除其他立克次体感染后确诊16例北亚蜱传斑点热(北亚热)病例。调查本地常住人口273人,血清抗体阳性率为15.8%,ＧＭＴ为13.8,其中病例组阳性率为18.0%(38/211),ＧＭＴ为14.6,对照组阳性率为8.1%(5/62),ＧＭＴ为9.1,男女阳性率分别为14.4%(19/132)…     

类鼻疽伯克霍尔德菌跨洲际流行株的同源性分析及历史溯源

目的 比较不同年代分离自澳大利亚、中国海南地区7株ST562型类鼻疽伯克霍尔德菌（类鼻疽伯克菌）的同源性,并分析其传播来源。方法 利用生物信息学方法提取ST562型类鼻疽伯克菌澳大利亚临床株MSHR5858、中国海南历史菌株350105基因组中Spe Ⅰ限制性酶切片段指纹谱、多位点可变数目串联重复序列多态性指纹（MLVA-4）等遗传特征,并与近年分离的5株海南ST562型临床株的脉冲场凝胶电泳（PFGE,Spe Ⅰ酶切）、MLVA-4分子分型结果相比较。同时分析MSHR5858与350105在基因组水平的共线性及同源性。结果 5株海南ST562型临床株的PFGE带型相同（相似度>97%）且与澳大利亚临床株MSHR5858的Spe Ⅰ限制性带型一致;澳洲临床株（MSHR5858）与海南历史菌株（350105）基因组中Spe Ⅰ限制片段数分别为31和34,其中31个片段的长度一致。5株海南ST562临床株的MLVA-4型别各不相同,但HPPH43（MLVA-4指纹谱：10,8,10,8）与MSHR5858（10,8,8,6）在2341 k、1788 k两个位点重复数相同;HK003（11,8,15,7）、HK061（11,8,17,7）与历史菌株350105（11,8,11,8）在此二位点重复数也相同。此外,350105与MSHR5858在基因组水平具有良好共线性,共有基因占绝大部分,提示来源一致。结论 分离的7株ST562型类鼻疽伯克菌（包括中国海南临床分离株、历史菌株及澳大利亚临床分离株）遗传特征一致,且ST562型近年流行株与历史菌株350105可能具有同源关系。     

蜡样芽胞杆菌快速检测及其毒力基因筛选

目的筛选蜡样芽胞杆菌鉴定基因和快速检测毒力基因。方法选择分离自食品和土壤的代表性蜡样芽胞杆菌共329株,聚合酶链式反应（PCR）方法检测gyrB和groEL基因的种特异性,检测毒力基因在菌株中的分布特征。 基于检测结果,用多重PCR检测方案快速检测蜡样芽胞杆菌鉴定基因及其毒力因子。结果在蜡样芽胞杆菌及其近缘芽胞杆菌中,除1株苏云金芽胞杆菌扩增阳性外,gyrB基因具有蜡样芽胞杆菌种特异性;而groEL基因在4种芽胞杆菌中均有扩增。 6种毒力基因nheA、entFM、bceT、hblC、cytK和ces的携带率分别为84.19%、79.64%、49.24%、47.72%、47.11%和1.52%。 选择nheA、hblC、entFM、ces、cytK和gyrB用于快速鉴定蜡样芽胞杆菌及其毒力基因,获得了双重PCR扩增体系（gyrB和cytK）与4重PCR扩增体系（nheA、hblC、entFM和ces）的最佳检测方案。结论筛选的蜡样芽胞杆菌鉴定基因和毒力基因能够全面、特异、简便、高效地检测蜡样芽胞杆菌,可为食品安全检测及快速诊断提供依据,在实际检验工作中具有良好的应用前景。     

16S rRNA基因序列分析在非典型菌株鉴定中的应用

目的：建立16SrRNA基因序列分析鉴定细菌的方法，评价其对常规方法不能鉴定菌株的鉴定效果。方法：选择细菌的16SrRNA基因为靶序列，在两端保守区设计引物，对三株生化、形态不典型菌株提取模板进行PCR扩增，PCR产物纯化后进行克隆，测序。结果：三株细菌的16SrRNA基因序列与Genbank比较，YC1序列与大肠杆菌相似性〉99％，YC2序列与肺炎克雷伯菌相似性〉99％，YC3序列与缓症链球菌相似性〉98％。结论：16SrRNA基因序列分析的方法可以较好地鉴定常规方法难以鉴定的不典型菌株。     

广东省大埔县斑点热群立克次体血清学调查

目的：调查大埔县健康人群及鼠、牛中斑点热群立克次体分布情况。方法：微量室温补体结合法。结果：在２８２名健康人群血清中，２９例西伯利亚立克次体抗体阳性，阳性率为１０２８％（２９／２８２）；１１例康氏立克次体抗体阳性，阳性率为３９０％（１１／２８２）；１６例小蛛立克次体阳性，阳性率为５６７％（１６／２８２）。３０份鼠血清，西伯利亚立克次体抗体阳性率为１３３３％（４／３０），康氏立克次体抗体阳性率为３３３％（１／３０），小蛛立克次体抗体阳性率为１０００％（３／３０）。２５份牛血清，西伯利亚立克次体抗体阳性率为８００％（２／２５），康氏立克次体抗体阳性率为４００％（１／２５），小蛛立克次体抗体阳性率为４００％（１／２５）。结论：广东省大埔县林区存在有斑点热的自然疫源地。