巢式PCR快速检测炭疽芽孢杆菌

目的：快速简便地检测炭疽芽孢杆菌。方法：细菌培养物经反复冻融，SDS，蛋白酶K和煮沸处理后，作为PCR模板，根据炭疽芽孢杆菌质粒pX01中水肿因子（EF）基因设计两对引物，采用巢式PCR（nestedPCR)扩增目的基因，结果：从炭疽芽孢杆菌模板中成功扩增出1247bp的特异片段，而未在炭疽芽孢杆菌无毒株，蜡样芽孢杆菌和枯草杆菌模板中扩增出相应条带；第一次扩增能检出的最低细菌量是10^3拷贝；经再次巢式PCR，扩增出208bp的特异片段，最低检出的细菌数为10个拷贝，敏感性提高了100倍。结论：巢式PCR是一种快速，特异，敏感的检测炭疽芽孢杆菌的方法。     

蜡状芽孢杆菌相关食物中毒研究进展

近年来蜡状芽孢杆菌(Bacillus cereus)已经成为一种新型食源性致病菌。B.cereus主要污染乳、乳制品、米面制品等,食用了被产毒素B.cereus污染的食物可能会造成摄食人群发生食物中毒,夏秋季高发。蜡状芽孢杆菌相关食物中毒的主要症状为呕吐和腹泻,这两种症状是由于其在生长过程中产生的呕吐毒素(环状肽,Cereulide)和腹泻毒素(5种肠毒素)所致。不同的菌株可产生不同的毒素,同一菌株在不同生长阶段也可产生不同毒素。通过分析蜡状芽孢杆菌食物中毒的原因、规律及其毒素作用机制,探讨预防该类食物中毒的措施。     

PCR方法快速检测炭疽芽孢杆菌

目的：快速检测炭疽芽孢杆菌。方法：质粒提取及PCR扩增。结果：用两对引物扩增，阳性样本可分别扩出两条877bp和1089bp左右的条带，阴林样本未扩增出。结论：所采用的方法可快速检测出环境中的炭疽芽孢杆菌，并可对其致病性进行评价。     

炭疽芽孢杆菌基因分型的研究进展

炭疽芽孢杆菌按Bergey分类属于杆菌科需氧芽孢杆菌属(Cohn1872)群Ⅰ中菌体在0．9μm以上的一簇。其中包括炭疽芽孢杆菌(Bacillus anthracis)、腊样芽孢杆菌(B．cereus)、蕈状芽孢杆菌(B．mycoides)、假真菌样芽孢杆菌(B．pseudomycoides)、苏云金芽孢杆菌(B．thuringiensis)和B．weihenstephanensis菌。Helgason认为B．anthracis是在1万～2万年间从B．cereus和B．thuringiensis菌种进化而来，并偶然获得2个质粒的菌种。     

苏云金芽孢杆菌杀蚊机制研究概况

从1976年起，人们就将目光转向苏云金芽孢杆菌（Bt）对蚊虫的研究和应用上。笔者简要描述了Bt主要的杀蚊晶体蛋白和杀蚊机制，并针对当前研究概况提出展望。随着杀虫晶体蛋白（ICPs）结构与功能研究的不断深入，必将不断丰富和完善ICPs分子作用机制，为进一步在分子水平上利用基因操作及蛋白质工程技术开发这一潜在有广阔应用前景的微生物杀蚊剂奠定基础。     

实时荧光定量PCR法检测人粪便中艰难梭状芽孢杆菌

目的摸索并明确实时荧光定量PCR技术检测人体肠道粪便中艰难梭状芽孢杆菌的方法。方法用16S rRNA real-time PCR方法检测喘息患儿与正常儿童肠道内梭状芽孢杆菌的数量。结果常规PCR扩增出艰难梭状芽孢杆菌16S rRNA部分基因片段长度为500 bp~750 bp,测序结果正确,符合预期,成功构建了艰难梭状芽孢杆菌实时荧光定量PCR中的标准品,生成的标准曲线r20.995;实时荧光定量PCR产物的溶解曲线为单峰,表明实时荧光定量PCR的特异性强。对待测80份样本分批进行荧光定量PCR检测,各组间标准品Ct值的变异系数(CV)为4.96%、3.45%、2.46%、2.43%和2.75%。80份儿童粪便样本中艰难梭状芽孢杆菌含量的对数值为3.70±2.71(copy/g湿便)。结论本研究表明,实时荧光定量PCR方法操作简便、灵敏度高、特异性强、自动化程度高,适用于人体粪便中艰难梭状芽孢杆菌含量的检测。     

3种不同方法对入境旧书中携带蜡样芽孢杆菌的检测分析

目的对一株从美国入境旧书中分离的菌株进行鉴定。方法从旧书表面采集样本分离获得一株菌株,分别采用VITEK生化鉴定、16s rDNA序列分析法及特异性引物PCR 3种方法进行鉴定。结果VITEK生化鉴定判定可能属于蜡样芽孢杆菌、苏云金芽孢杆菌或蕈状芽孢杆菌的一种。16s rDNA通用引物PCR检测结果显示该菌株与芽孢杆属同源性最高达99%。特异性引物PCR检测结果判定为蜡样芽孢杆菌。结论初筛和特异性检测方法相结合,可以有效的对入境货物携带的致病微生物进行鉴定。     

用IS6110限制性片段长度多态性方法分析中国南方部队结核分支杆菌DNA指纹特征

目的：分析南方部队结核病患者和当地患者中结核分支杆菌分离株DNA指纹特征，探讨南方部队结核病的分子流行病学特征。方法：用限制性内切酶PvuⅡ消化结核分支杆菌DNA，后用琼脂糖凝胶电泳，再用Southern免疫转印，用[α^32P]-dCTP标记的DNA IS6110序列中的245bp片段作探针，进行杂交后得到限制性片段长度多态性图谱，结合一般流行病学资料加以分析比较。结果：共检测185株结核分支杆菌分离株。检测菌株的IS6110拷贝数范围为1～22。部队患者和当地患者的IS6110拷贝数分布差异无显著姓。部队患者结核菌分离株的IS6110拷贝数主要集中在6～20个，当地分离株主要集中在7～20个；全部菌株指纹特征分成8个组，部队分离株和当地分离株均主要集中在Ⅰ、Ⅱ、Ⅲ 3个组里。耐药菌株指纹特征在各组中的分布与敏感菌株差异有显著性；患者是否接种卡介苗在各组中的分布差异无显著性。结论：南方部队患者与当地患者结核菌分离株在遗传关系上较接近，在基因水平上相关程度较强。提示部队结核病的发生与当地结核分支杆菌菌株的传播密切相关。     

苏云金芽孢杆菌杀蚊蛋白基因cry11Aa的克隆与表达

目的 克隆苏云金芽孢杆菌以色列亚种的杀蚊毒素蛋白基因cry11 Aa，并使其在酵母双杂交系统受体菌Saccharomyces cerevisiae L40中获得表达。方法 应用聚合酶链反应扩增获得cry11 An基因，构建表达载体pHybLex／Zeo-cry11 Aa，转化到酵母菌Saccharomyces cerevisiae L40。结果 用CryllAa抗体和LexA抗体进行的Western blot免疫杂交表明cry11Aa基因在酵母菌L40中成功表达，生成了Cry11 Aa-LexA融合蛋白。结论 成功地克隆、表达了cry11Aa基因，为进一步利用酵母双杂交系统寻找与毒素蛋白Cry11Aa特异性结合的蚊幼中肠受体蛋白，揭示苏云金芽孢杆菌毒杀蚊虫的分子生物学机制奠定了基础。     

微生物菌剂中5种芽孢杆菌实时荧光PCR鉴定

目的:建立一种快速、准确鉴定环保微生物菌剂中常用5种芽孢杆菌的方法。方法:根据rpoB和gyrB基因序列分别设计扩增5种菌的特异性引物及Taqman探针,并进行反应条件优化。结果:5种芽孢杆菌均产生特异扩增信号,最低检出浓度为:枯草芽孢杆菌24 cfu/ml、地衣芽孢杆菌35 cfu/ml、巨大芽孢杆菌50 cfu/ml、短小芽孢杆菌41 cfu/ml、环状芽孢杆菌21 cfu/ml。结论:研究建立的实时荧光PCR鉴定方法具有良好的重复性和准确性,可用于微生物菌剂中5种常用芽孢杆菌的快速鉴定,具有很好的推广应用前景。     

沙门菌基因间共有重复序列-PCR分子分型方法的优化及应用初探

目的优化沙门菌基因间共有重复序列-PCR(ERIC-PCR)分子分型方法,分析沙门菌标准菌株及流行分离株基因组DNA ERIC-PCR指纹图谱。方法提取鼠伤寒沙门菌基因组DNA作为模板,根据ERIC核心片段设计引物,运用ERIC-PCR技术扩增沙门菌基因组中的靶序列,PCR产物经琼脂糖凝胶电泳后用凝胶成像分析仪对图谱进行观察分析。反应体系中模板量、Mg2+浓度、引物浓度和退火温度的优化实验则采用对优化项目设置梯度、其它条件不变的方法进行。以优化好的ERIC-PCR方法对16株沙门菌及1株大肠杆菌进行分析。结果在总体积为25μl的反应体系中,选择DNA模板量为100ng/25μl,Mg2+浓度为2.0mmol/L,引物ERIC1R、ERIC2浓度分别为0.4μmol/L,退火温度为52℃时,扩增产物的电泳图谱条带最清晰完整。不同血清群、型和来源的沙门菌株间基因组DNA ERIC-PCR指纹图谱带型差异较大,可在250～5000bp范围内出现3～13条条带,并在250bp处出现一条特征条带。结论优化了沙门菌ERIC-PCR的重要反应条件。ERIC-PCR法能区分不同来源的沙门菌,可用于沙门菌病的流行病学调查和同源性追踪,弥补传统分型方法的不足。     

运用UP-PCR技术进行食源性感染常见致病菌Hsp60基因的扩增与酶切鉴定

目的 扩增、鉴定食源性感染常见致病菌Hsp60基因。方法 选取13种食源性感染常见致病菌，采用UP-PCR法扩增其Hsp60基因，并对PCR产物进行酶切鉴定分析。结果 13种食源性感染常见致病菌均可以扩增出约1．1KB的基因片段，蜡样芽孢杆菌PCR产物经HindⅢ酶切后形成约684bp和470bp片段，小肠结肠炎耶尔森菌PCR产物经BglⅡ酶切后形成861bp和296bp片段。结论 UP-PCR技术能同时扩增出食源性感染常见致病菌Hsp60基因，为进一步进行致病菌的种属鉴定和分型研究奠定基础。     

Development of a High-Resolution Melting-Based Approach for Efficient Differentiation Among Bacillus cereus Group Isolates

Abstract Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.     

Isolation and characterization of Bacillus cereus-like bacteria from faecal samples from greenhouse workers who are using Bacillus thuringiensis-based insecticides

OBJECTIVES: Since the discovery of the insecticidal activity of Bacillus thuringiensis at the beginning of the twentieth century, this bacterium has been used increasingly against various insect pests. In spite of the extensive use of B. thuringiensis, only sporadic clinical case reports have been published. In recent years, the close relationship between B. thuringiensis and the human pathogen Bacillus cereus has been confirmed. In practice, only the insecticidal activity of B. thuringiensis distinguishes the two species. However, both species are composed of thousands of isolates with varying potential for causing adverse effects in humans. The aim of this study was to employ molecular biology methods for assessment of occupational exposure to B. thuringiensis-based biopesticides by determination of specific genetic information including plasmid profiles and random amplified polymorphic DNA (RAPD). METHODS: Faecal samples from 12 persons, working in Danish greenhouses, were collected for microbial analysis. Seven persons were using B. thuringiensis-based insecticides, whereas five persons were employed at greenhouses that did not use B. thuringiensis. The bacteria were isolated on B. cereus-specific solid substrate, and colonies were further identified using the polymerase chain reaction (PCR). The PCR method was used for the identification of the enterotoxin genes HblA and BceT. The expression of enterotoxins was detected with two commercial serological kits. Primers specific for 16S-23S spacer region were used to identify the bacteria as members of the B. cereus group. Several primers towards insecticidal genes have been used in order to further characterize the isolates as subspecies of B. thuringiensis. RESULTS: Two faecal samples from the B. thuringiensis-exposed greenhouse workers were positive for B. cereus-like bacteria. One isolate displayed intracellular crystalline inclusions characteristic of B. thuringiensis, production of and genes for B. cereus enterotoxins and it was PCR-positive for an insecticidal toxin primer set. RAPD profiles of the faecal isolate were identical to that of strains isolated from a commercial product. CONCLUSIONS: The methods applied have verified that the faecal isolate was identical to the B. thuringiensis isolate found in the biopesticide used. This is the first reported case of isolation of a bacterial biopesticide from human faeces. The biopesticide was shown to harbour and express enterotoxin genes. However, there is no evidence that this caused any adverse effects to the person from whom these bacteria were isolated.     

医院感染耐亚胺培南铜绿假单胞菌与oprD_2基因相关的ERIC分型研究

目的 探究耐亚胺培南铜绿假单胞菌(IRPA)的oprD2基因缺失在天津地区3所三甲医院内的分布以及该菌的克隆流行情况.方法 采用聚合酶链反应(PCR)方法检测60株IRPA的oprD2基因缺失情况,并以基于肠杆菌科基因间重复一致序列PCR(ERIC-PCR)方法对其中的54株进行分子生物学分型.结果 60株IRPA中有38株oprD2基因缺失.且3所医院的oprD2基因缺失率差异无统计学意义(P＞0.05),54株IRPA用ERICPCR方法分为30型.结论 IRPA的oprD2基因缺失在天津地区的分布差异无统计学意义,且提示尚有其他原因造成亚胺堵南/西司他丁耐药株流行;个别科室内的IRPA存在克隆流行趋势,应注意监控IRPA在医院内的暴发流行.     

PCR-based characterization of Yersinia enterocolitica: comparison with biotyping and serotyping.

PCR-based DNA fingerprinting was used to characterize 48 clinical isolates of Yersinia enterocolitica. The samples were examined by random amplified polymorphic DNA (RAPD-PCR) and inter-repeat PCR (IR-PCR). IR-PCR with two enterobacterial repetitive intergenic consensus primers resulted in patterns which were poorly discriminated; 2 of 11 arbitrary primers (RAPD-PCR) provided sufficient discriminatory power. In comparisons with serotyping and biotyping, RAPD-fingerprinting was the most discriminatory technique and may therefore be a valuable epidemiological tool for the study of Y. enterocolitica infections.     

致病性大肠埃希菌高致病性毒力岛irp2基因序列的扩增与分析

目的探讨致病性大肠埃希菌（EPEC）中高致病性毒力岛（HPI）irp2基因与致病性的关系。方法根据Genebank中irp2基因设计特异性引物，应用聚合酶链反应（PCR）技术扩增基因全长，克隆入pUCm-T载体，并通过酶切和测序鉴定。结果160株EPEC中有16株（10．00％）扩增出278bp基因片段，该基因片段与GeneBank中公布的耶尔森菌HPI irp2基因的同源性高达97％以上。结论部分EPEC具有耶尔森菌毒力岛HPI基因序列，该毒力岛可能与EPEC的致病性有一定相关性。     

Intraspecific comparative genomics of Candida albicans mitochondria reveals non-coding regions under neutral evolution

The opportunistic fungal pathogen Candida albicans causes serious hematogenic hospital acquired candidiasis with worldwide impact on public health. Because of its importance as a nosocomial etiologic agent, C. albicans genome has been largely studied to identify intraspecific variation and several typing methods have been developed to distinguish closely related strains. Mitochondrial DNA can be useful for this purpose because, as compared to nuclear DNA, its higher mutational load and evolutionary rate readily reveals microvariants. Accordingly, we sequenced and assembled, with 8-fold coverage, the mitochondrial genomes of two C. albicans clinical isolates (L296 and L757) and compared these sequences with the genome sequence of reference strain SC5314. The genome alignment of 33,928 positions revealed 372 polymorphic sites being 230 in coding and 142 in non-coding regions. Three intergenic regions located between genes tRNAGly/COX1, NAD3/COB and ssurRNA/NAD4L, named IG1, IG2 and IG3, respectively, which showed high number of neutral substitutions, were amplified and sequenced from 18 clinical isolates from different locations in Latin America and 2 ATCC standard C. albicans strains. High variability of sequence and size were observed, ranging up to 56 bp size difference and phylogenies based on IG1, IG2 and IG3 revealed three groups. Insertions of up to 49 bp were observed exclusively in Argentinean strains relative to the other sequences which could suggest clustering by geographical polymorphism. Because of neutral evolution, high variability, easy isolation by PCR and full length sequencing these mitochondrial intergenic regions can contribute with a novel perspective in molecular studies of C. albicans isolates, complementing well established multilocus sequence typing methods.     

大肠杆菌O157特异基因的PCR检测方法

目的：建立一种灵敏、特异的检测肠出血性大肠杆菌O157的PCR方法。方法：针对大肠杆菌O157特异性基因rfbO157设计引物，扩增-497bp的O157标识序列。结果：应用该PCR反应体系，对4株O157菌株和14株非O157菌株扩增结果表明，O157菌株均扩增出预期的497bp片段，14株非O157菌株则为阴性。纯菌液检测灵敏度为60cfu／PCR反应，模拟混合菌液检测灵敏度为400cfu／PCR反应。结论：该方法用于肠出血性大肠杆菌O157的检测鉴定简便、快捷，具有很高的特异性和灵敏度，为O157的临床辅助诊断提供了有力手段。