梅毒螺旋体外膜蛋白Gpd的基因型分析及其重组蛋白的免疫原性鉴定

目的　构建梅毒螺旋体 (Treponemapallidum ,Tp)外膜蛋白Gpd基因的原核表达载体 ,检测其表达产物的免疫原性 ,并比较梅毒螺旋体各菌株Gpd基因序列的同源度。方法　从TpNichols株基因组模板中PCR扩增Gpd基因 ,与GenBank登录的序列做blast比较 ,定向克隆构建原核表达重组体pET2 8b( + ) -Gpd ,转入大肠杆菌ril表达菌 ,SDS -PAGE分析重组蛋白的表达 ,Western -blot检测重组蛋白的免疫原性。结果　载体上所连目的基因片段序列与GenBank登录的Nichols株Gpd基因序列完全一致 ,同其他病原性密螺旋体菌株登陆序列比较同源度为 98%～ 1 0 0 %。SDS -PAGE检测诱导产物显示有一Mr约为 41kDa的特异蛋白带 ,免疫印迹技术检测其能与梅毒阳性标准血清反应。结论　Tp原核表达重组体pET2 8b( + ) -Gpd成功构建 ,且能够在ril表达菌中融合表达 ,为进一步研究该蛋白的生物学功能奠定了一定的实验基础。     

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities and specificities of 94-100% were achieved using two real-time PCR platforms, the Rotor-Gene and the iCycler. The assay can be completed within 2 h, enabling reporting in <8 h. This fast and robust assay is suitable for implementation in routine laboratories for diagnosing primary syphilis.     

Environmental effects on the growth and proteolysis of Treponema denticola ATCC 33520

The effect of pH, redox potential, O₂ and H₂ on the growth and proteolytic activity of Treponema denticola ATCC 33520 was studied in a chemostat at different growth rates. The peptidase and protease activities were estimated using different amido-methyl coumarin derivatives and azocasein. The maximum growth rate of T. denticola ATCC 33520 was 0.14 h⁻¹. Reduction of the growth rate of T. denticola by 50–60% gave: an increase in cell mass of 150–200%, a higher acetogenesis and a shift of the pH optimum. The protease and phenylalanine peptidase activities seemed to be of greater importance for the growth of T. denticola ATCC 33520 than the rather low arginine and proline peptidase activities. The redox potential (E_h) played a secondary role. At microaerophilic conditions with 1–5% O₂, the cultures maintained a redox potential below −311 mV and an optimal acetogenesis. The presence of H? induced a marked growth stimulation of T. denticola ATCC 33520. It is concluded that the cell mass and proteolytic activity of T. denticola ATCC 33520 are modulated by the growth rate and the pH and to a lesser extend by the redox potential and presence of O₂. Stagnation of the exudate-flow influences these factors and will lead to an increase of the spirochetal population and proteolysis in the periodontal pocket.     

PCR检测龈下菌斑中齿垢密螺旋体与牙周组织破坏的关系

目的　建立龈下菌斑标本中齿垢密螺旋体PCR临床快速检测方法 ,了解齿垢密螺旋体感染与慢性牙周炎牙周组织破坏程度之间的关系。方法　 81例慢性牙周炎患者每例采取 2个不同患牙牙位的龈下菌斑标本 ,用终浓度为 1%的TritonX 10 0 10 0℃水浴 10min处理标本以制备DNA模板 ,用PCR检测标本中齿垢密螺旋体特有的相对分子质量 (Mr)为 5 3× 10 3 外膜蛋白基因 (tdpA)扩增片段。结果　 6 7例患者 ( 82 .7% )的 2份龈下菌斑标本齿垢密螺旋体tdpAPCR均为阳性 ,9例患者( 11.1% )的其中 1份龈下菌斑标本可见目的扩增片段 ,5例患者 ( 6 .2 % )的 2份龈下菌斑标本PCR均为阴性 ,16 2例龈下菌斑PCR检测总阳性率为 88.3% ( 143 16 2 )。牙槽骨吸收 >2 3和牙周袋深度≥7mm患牙龈下标本的PCR阳性率较高 (P <0 .0 5 ) ,牙龈指数与PCR阳性率无明显关系 (P >0 .0 5 )。结论　PCR检测tdpA基因可用于慢性牙周炎龈下标本中齿垢密螺旋体的临床快速诊断 ,齿垢密螺旋体感染与牙周组织破坏密切相关。     

Construction of the Eukaryotic Expression Vector for Outer Membrane Protein Tp92 from Treponema pallidum and Its Preliminary Study on the Immune Responses in New Zealand Rabbits

Treponema pallidum subsp. pallidum is the causative a gents of syphilis. Presently, the annual infection rate ofdomestic and international syphilis remains rather high [1,2]. Apart from the serious nature of the disease itself, anumber of studies sugg…     

梅毒螺旋体抗体筛查及其临床意义

目的研究梅毒螺旋体的感染状况，探讨梅毒螺旋体抗体筛查的现实意义。方法采用酶联免疫分析法（ELIsA）检测梅毒螺旋体抗体，阳性者再使用甲苯胺红不加热血清试验（TRusT）和梅毒螺旋体明胶凝集试验（TPPA）做进一步检测。结果5998例住院患者ELISA法共检测出阳性86例，TPPA法检测出阳性78例，TRUST法检测出阳性22例。高龄（〉70岁）患者的阳性检出率高于其它年龄组。结论住院患者常规筛查梅毒螺旋体抗体可有效检测出隐性梅毒患者，不仅可以早诊断、早治疗，减少医疗纠纷的发生，也可以预防院内交叉感染。     

疑似梅毒患者血液47KDa和15KDa膜免疫原基因的测定

目的研究巢式聚合酶链反应(PCR)在排除梅毒血清学试验假阳性中的作用。方法用巢式PCR和常规PCR扩增90例疑似梅毒患者全血中梅毒螺旋体(TP)的47KDa和15KDa的膜免疫原基因,并与血清学方法(RPR,TPPA)的检验结果作比较。结果巢式PCR检测TP的敏感性和特异性分别为93.33%和96.67%,两者符合率为96.67%;两种PCR方法检测结果差异有统计学意义(P<0.01),巢式PCR敏感性高于常规PCR。结论巢式PCR检测全血中TP较常规PCR灵敏,通过检测47KDa和15KDa的膜免疫原基因,有助于排除梅毒血清学试验假阳性,提高了梅毒诊断的准确率。     

Current status of syphilis in pregnant women in Japan

We examined the current status of syphilis-infected pregnant Japanese women, according to the results of syphilis screening and confirmation tests of women who gave birth in Japan between October, 2015 and March, 2016. We requested 2458 obstetrical facilities to provide information of syphilis screening tests and 78.1% of them responded. Considering the response rate and the rate of implementation of confirmation tests, the number of syphilis-infected pregnant Japanese women was estimated to be 250 (1/4022) per year.     

5334例住院患者HBsAg、抗-HCV、抗-HIV1/2和TP-Ab的检测结果分析

目的 检测5334例住院患者HBsAg、抗-HCV、抗-HIV1/2和TP-Ab并分析检测结果.方法 采用ELISA法检测5334例住院患者HBsAg、抗-HCV、抗-HIV1/2和TP-Ab阳性情况,并分析HBsAg、抗-HCV、抗-HIV1/2和TP-Ab检测阳性者性别和年龄分布.结果 5334例住院患者HBsAg阳性率最高(7.56％),抗-HIV1/2阳性率最低(0.52％);男性患者HBsAg、抗-HCV、TP-Ab检测阳性率与女性患者比较,差异无统计学意义(P＞0.05);男性患者抗-HIV1/2检测阳性率显著高于女性患者,差异具有统计学意义(P＜0.05);成年组患者HBsAg和抗-HCV阳性率均显著高于青少年组,差异具有统计学意义(P＜0.05);老年组患者HBsAg、抗-HCV和TP-Ab阳性率均显著高于青少年组,差异具有统计学意义(P＜0.05);老年组患者HBsAg、TP-Ab阳性率均显著高于成年组,差异具有统计学意义(P＜0.05);不同年龄组患者抗-HIV1/2阳性率比较,差异无统计学意义(P＞0.05).结论 住院患者乙型肝炎感染率较高,艾滋病感染存在性别分布差异,乙肝、丙肝和梅毒感染在年龄分布上存在差异.