目的 研究蜡样芽胞杆菌毒力基因在不同标本中的分布特征.方法 分离自环境监测标本(米粉、奶粉、土壤)和疾病相关标本(米饭、凉皮、眼内炎和肿瘤患者)的蜡样杆菌333株,PCR扩增蜡样杆菌11个毒力基因,包括溶血性BL基因(hblC、hblD、hblA、hblB)、非溶血性基因(nheA、nheB、nheC)、肠毒素FM基因和T基因(entFM、bceT)、细胞毒素K基因(cytK)和呕吐毒素相关基因(ces),统计不同标本菌株中毒力基因的携带数目和各毒力基因的携带率,方差分析和卡方检验比较毒力基因在不同标本中,尤其是在疾病相关标本与环境监测标本中的携带差异.结果 研究菌株携带毒力基因平均数目为5.97个,88.29％的菌株携带至少3个毒力基因,12.31％的菌株携带除ces外的所有基因.标本携带毒力基因数目从高到低依次为患者(8.75)、凉皮(8.20)、米饭(7.13)、土壤(6.22)、米粉(5.78)和奶粉(5.71).患者、凉皮分别与奶粉的基因数目两两比较有统计学差异显著性.菌株各毒力基因的携带率从高到低依次为非溶血性基因(89.19％)、entFM基因(79.88％)、bceT基因(49.85％)、溶血性BL基因(48.35％)、ctyK基因(47.75％)和ces基因(1.50％).溶血性基因在疾病相关标本中的携带率比环境监测标本高(x2 =8.230,P＜0.01),其余基因携带率则在两类标本中无统计学差异.环境监测标本中,土壤的溶血性基因携带率高于米粉和奶粉(x2=15.071,P＜0.01),非溶血性基因和entFM基因的携带率则低于后两者,检验值分别为(x2=9.603,P＜0.05)和(x2=21.634,P＜0.01).结论 蜡样杆菌毒力基因在不同标本中的分布,尤其是毒力基因数目和溶血性基因在疾病相关标本中较高的携带特点,为研究蜡样杆菌的致病性提供了一定的参考依据,具有一定的临床意义.

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(gryB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.

Objective To type Yersinia pestis isolated from Gansu Province,and to study the trend of diffreent strains in different administrative regions and different years.Methods Totally 193 strains were enrolled in this study,including 9 strains of Ganning Dauricus type,18 strains of Aerjin type,45 strains of Qilian type and 121 strains of Qingzang type.These strains were genotyped by clustered regularly interspaced short palindromic repeats (CRISPR),and genotypes were named according to international standard.Genotyping by CRISPR in different administrative regions and different years of Gansu Province was explored.Results Two clusters (Ca7 and Cb4),including four genotypes (genotypes 7,22,24 and 26) were classified by CRISPR.From the point of view of origin,genotype 24 was the main genotype in Akesai 36.36％ (16/44),Subei 36.17％ (17/47),Yumen 50.00％ (5/10) and Su'nan 38.67％ (29/75);the main genotype of Xiahe and Huining was genotype 26 (4/7);the main genotype of Shandan was genotype 22 (1/1).From the point of view of time,the main genotype of Yersiniapestis in Gansu Province during the years of 1960-1969,1970-1979 and 1980-1989 was genotype 26 [53.33％ (8/15),60.00％ (6/10) and 48.28％ (14/29)];the main genotype was genotype 22 [40.91％ (18/44)] during the years of 1990-1999;and the main genotype was genotype 24 [43.16％ (41/95)] during the years of 2000-2009.Conclusion Four genotypes of Yersiniapestis in Gansu Province are quite different in different administrative regions and different years.

Objective Using quantitative real-time PCR to establish a rapid specific genetic diagnostic technique for Yersinia pestis.Methods ①Four sets of specific probes and primers were designed,which targeted to chromosome genes of YPO0392,YPO1094,YPO2087 and YPO2090,respectively.②The probes and primers were tested for stability and specificity with 40 strains of Yersinia pestis and 47 strains of non-Yersinia pestis of different sources in Yunnan.③Eight positive DNA in Yulong,Yunnan,were tested with the screened probes and primers.Results ①Two sets probes and primers were selected,they were targeting YPO0392 and YPO1094,respectively.②The results were all positive of the eight positive DNA samples tested.Conclusion Two sets of primers and probes are selected for rapid specific diagnosis of Yersinia pestis.

目的 通过比较蜡样芽胞杆菌(以下简称蜡样杆菌)脉冲场凝胶电泳(PFGE)技术方法的影响因素,优化蜡样杆菌PFGE分型方法.方法 在细菌收集、细胞裂解、胶块制备和电泳参数等环节设立对照,选取3株蜡样杆菌比较不同实验方案的电泳图谱结果.细菌收集设立LB固体培养基和液体培养基集菌法;细胞裂解设立灌制胶块前溶菌酶孵育组与未孵育组,设立单独溶菌酶裂解组和溶菌酶与溶葡萄球菌酶联合裂解组;细菌的胶块内裂解设立溶菌酶不同浓度和不同作用时间(2、4和16h)实验组;选取22株蜡样杆菌对3组电泳参数进行分析评价.结果 LB固体培养基集菌法与液体集菌法相比,获得了同样清晰或更清晰的条带.灌制胶块前溶菌酶与菌悬液进行一定时间的孵育可明显改善条带的清晰度.溶菌酶对胶块内细菌的裂解,4h为最佳裂解时间.溶葡萄球菌酶与溶菌酶联合使用未见对细菌裂解有改善作用.3组电泳参数(EP)的图谱差异有统计学意义(P＜0.05),EP-a的相似性系数最小,为最佳电泳参数.结论 试验确定了最佳蜡样杆菌PFGE实验方案,简化和优化了目前已有的蜡样杆菌PFGE实验方法,可应用于蜡样杆菌引起的暴发疫情分析及食源性病原菌的实验室分子分型研究.

Objective To analyze the genotypic features of Cronobacter spp.isolated in China using multiple-locus variable number tandem repeat analysis(MLVA).Methods 62 isolates of Cronobacter spp.were obtained and amplified by PCR using four variable number of tandem repeat(VNTR) loci primers followed with further analysis of capillary electrophoresis and sequencing.A dendrogram was constructed by BioNumerics.Results Out of 62 strains of Cronobacter spp.isolated in China,8 clusters and 29 MLVA types were identified,and 43.5% of stains were classified as cluster II.Also multiple MLVA types were found to be from the same region and the same type from different regions.Conclusions Cronobacter spp.isolated in China are genetic polymorphic.MLVA has a higher discriminative typing capability,while the VNTR loci need to be optimized for Chinese isolates.

To investigate the function for receptor-binding of the tail fiber protein gp44 of Vibrio cholerae typing phage VP3 and to test whether gp44 is the site of interaction with its receptor on V.cholerae cell surface,gp44 was cloned and expressed with 6-His tag added to its N-terminus. The purified His-gp44 in solution was then incubated with the strain susceptible or resistant to the lytic action of VP3, its whether it wuld bind the susceptible or resistant strains was observed by fluorescent microscopy and laser scanning co-focal microscopy. Meanwhile,the sequence of the wav gene cluster wav the wild type V.cholerae with natural resistant to VP3 was DCR,amplified,and sequcnced.It was found that His-gp44 located on the surface of both susceptible and resistant stains,but not on the surface of Escherichiawti. When one of the genes in gene cluster wav was deleted,the susceptible mutant showed no His-gp44 around its cell wall. The sequence of the gene cluster wav showed only one single base difference in susceptible and resistant strains to VP3. It is concluded from the above mentioned observations that the tail fiber protein gp44 of V. cholerae typing phage VP3 can bind specifically with the surface receptors on V. cholerae.

猪链球菌2型是重要的人畜共患病病原,在世界范围内广泛流行,可以引起人感染猪链球菌病暴发流行。猪链球菌2型粘附能力是细菌毒力作用的一个重要表现。猪链球菌2型的粘附过程是细菌和上皮细胞以及组织相互作用的过程,其中纤连蛋白结合蛋白和纤连蛋白的结合介导细菌粘附到宿主细胞上。另外猪链球菌2型还与巨噬细胞相互作用,荚膜对粘附也产生影响。

Objective:The phage-biotyping scheme can differentiate the potential pathogenicity of different Vibrio cholerae strains.VP3 is one of the five typing phages.To complement the annotation of the VP3 genome,the protein composition was analyzed.Methods:On the basis of complete genome sequencing and bioinformation analysis,the structure proteins of the purified mature VP3 particles were separated and identified using two-dimension electrophoresis and MS.Results:20 protein spots were separated and 10 of them were identified by MALDI-MS/MS,which corresponded to four proteins of VP3 and four proteins of V.cholerae.Conclusion:The results indicate that there was high similarity between the structure protein compositions of VP3 and T7.The host proteins purified with the phage particles may play some roles in the transduction of VP3.

一、炭疽芽孢杆菌在自然界的生存及致病作用概述 炭疽(anthrax)是由炭疽芽孢杆菌(bacillus anthracis)引起的一种人兽共患病,主要发生在牛及其他食草哺乳动物,患者通过接触被感染的动物和存在于空气、土壤中的炭疽杆菌芽孢发病,一般不形成人与人的直接传播.