应用实时荧光PCR检测致病性蜡样芽孢杆菌

目的:利用SYBRGreenⅠ染料能与双链DNA结合发出荧光的特点,建立一种用来检测致病性蜡样芽孢杆菌(B.cereus)的实时PCR方法。方法:对致病性蜡样芽孢杆菌致病相关基因cereolysinAB基因序列进行分析,设计1对特异引物,通过对SYBRGreenⅠ实时PCR反应条件的优化,实现对致病性蜡样芽孢杆菌的检测。结果:该方法具有较强的灵敏性及特异性,能够对致病性蜡样芽孢杆菌进行有效检测,其最低检出限可达8CFU/mL。结论:利用该技术检测蜡样芽孢杆菌,较常规的生化检测方法具有省时省力的特点,且灵敏性较高,具有实际应用价值。     

牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。     

蜡样芽孢杆菌检测方法的研究进展

蜡样芽胞杆菌(Bacillus cereus)是革兰氏阳性芽孢杆菌,广泛存在于自然环境中,对不良环境有较强的抵抗力,是一种人畜共患的食源性条件致病菌.蜡样芽孢杆菌能够产生多种毒素,这些毒素决定了其致病性.因此,建立快速、准确的蜡样芽孢杆菌检测方法对疾病的诊断和发病后的及时治疗至关重要.本文对近年来检测蜡样芽孢杆菌的菌体...     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

蜡样芽孢杆菌主要毒素及检测方法的研究进展

蜡样芽孢杆菌(Bacillus cereus)是一种普遍存在的食源性病原菌,可造成腹泻和呕吐两种类型的食物中毒和其他类型的疾病根据蜡样芽孢杆菌引起食物中毒的症状,其毒素可分为致腹泻型肠毒素和致呕吐型肠毒素,其中溶血性肠毒素BL、非溶血性肠毒素和细胞毒素K是引起腹泻型食物中毒的3种主要毒素,cereulide为致呕吐型肠...     

蜡样芽孢杆菌肠毒素的研究动态

通常认为蜡样芽孢杆菌(Bacillus cereus)是一种条件致病菌。临床上偶可致脓肿、脑膜炎、肺炎、骨髓炎、心内膜炎及败血症等,但以引起食物中毒最为重要。自1950年以来,Hauge曾多次从食物中毒的标本中分离出蜡样芽孢杆     

炭疽芽孢杆菌检测鉴定技术研究进展

炭疽芽孢杆菌的感染,无论是自然感染,还是作为生物恐怖和生物战的手段。快速检验和鉴定疽芽孢杆菌是最为关键的,只有正确识别生物战剂的种类,才能为正确实施防治措施指明方向。本文讨论了炭疽芽孢杆菌的检验鉴定技术,包括常规的分析培养,免疫学技术,核酸分析技术,生物传感器,基因芯片技术的应用和新诊断分子,如肽核酸与适配子的应用。     

养殖中华鳖蜡样芽孢杆菌的分离、鉴定和致病性研究

2016年夏杭州某中华鳖(Pelodiscus sinensis)养殖场出现大量发病, 病鳖呈厌食、反应迟钝、四肢无力、池边独游、濒死前不停摇头等病症, 死亡率20%以上。用营养琼脂从典型症状濒死中华鳖分离到革兰阳性短杆状, 芽孢近中生或中生的分菌株; API 50 CHB对分离菌株T91-1鉴定结果显示与蜡样芽孢杆菌(Bacillus cereus)参考菌株ATCC 14579 97.4%相似; T91-116S rDNA和gyrB基因序列与ATCC 14579相似度为99%, 确认T91-1为蜡样芽孢杆菌。T91-1在羊血平板呈典型β溶血, 牛奶平板上出现明显透明圈, 表明存在溶血性和胞外蛋白酶类毒素。用T91-1肌肉注射感染健康中华鳖LD₅₀为4.91×105CFU/ind., 死亡鳖出现与自然发病鳖类似症状; 感染发病中华鳖血和肝印/涂片瑞氏-姬姆萨染色可见组织间隙有大量芽孢杆菌。自然发病鳖肾、肝、肺、心病理切片可见大量芽孢杆菌, 病鳖血管扩张充血, 嗜酸性粒细胞浸润。T91-1灌胃ICR乳鼠3d后可全部死亡, 死亡鼠呈皮下出血等症状; T91-1腹腔注射和灌胃ICR 4周龄小鼠12—24h可出现迟钝、厌食等症状, 1周内未出现死亡。上述结果表明蜡样芽孢杆菌对中华鳖有很强毒力, 对小鼠有较强肠毒性, 是引起本次杭州中华鳖暴发性疾病的病原。     

蜡样芽孢杆菌ATCC14579核黄素操纵子的克隆及在枯草芽孢杆菌中的表达

利用BLAST从B．cereus ATCC14579的基因组中找到一段与枯草芽孢杆茵核黄素操纵子具有较高相似性的4．6kb大小的基因组DNA片段,该片段中含有完整的核黄素操纵子。该操纵子结构基因的编码产物的氨基酸序列与枯草芽孢杆菌核黄素操纵子相应结构基因的编码产物的氨基酸序列具有99％的同源性。该片段被克隆到大肠杆茵一枯草芽孢杆茵穿梭载体pHP13M中。表达分析的结果表明B．cereus ATCC14579核黄素操纵子可在大肠杆茵和枯草芽孢杆菌中表达。利用PCR方法用来自枯草杆菌的sac B基因的启动子替换B．cereus ATCC14579核黄素操纵子原有的启动子使其更好表达。替换启动子后的核黄素操纵子在本文使用的发酵条件下有较好的表达,核黄素产量从39．5mg／L增加到61．7mg/L.     

草莓中蜡样芽孢杆菌的VITEK快速检测

采用形态、生理生化以及生物分子等多种手段开展了草莓中芽孢杆菌的鉴定研究,结果确认草莓携带的芽孢菌为蜡样芽孢杆菌。实验表明了VITEK微生物自动鉴定系统可快速检测草莓中的芽孢菌,结果可靠,为开发草莓杀菌抑菌技术提供了参考。     

利用ERIC-PCR对苏云金芽胞杆菌与蜡状芽胞杆菌进行鉴定的研究

为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。     

PCR法快速鉴定眼源性蜡样芽胞杆菌

目的建立一种快速、灵敏、特异的眼源性蜡样芽胞杆菌PCR检测方法,为蜡样芽胞杆菌性眼内炎患者的快速诊断提供依据。方法选择编码蜡样芽胞杆菌细胞毒素的cytK为靶基因设计引物,建立检测眼源性蜡样芽胞杆菌PCR;PCR产物用琼脂糖凝胶电泳鉴定,基因序列与GenBank比对验证扩增产物;将计数过的5株蜡样芽胞杆菌菌悬液,梯度稀释后分别提取DNA进行PCR扩增,确定检测方法的灵敏度;分别用眼部常见感染菌金黄色葡萄球菌、表皮葡萄球菌、甲型溶血性链球菌、化脓性链球菌、藤黄微球菌、铜绿假单胞菌、大肠埃希菌、普通变形杆菌和白假丝酵母菌以及枯草芽胞杆菌DNA进行特异性试验;进一步将该方法应用到人工污染致病蜡样芽胞杆菌的房水标本中,并分析其灵敏度。结果5株分离自眼内炎患者标本中的蜡样芽胞杆菌均扩增出360bp左右的DNA片段,测序结果与GenBank比对一致;该法检测在5h内完成,方法灵敏度达7．5～15．0CFU／mL;其他菌株检测未出现非特异性扩增;对模拟感染房水标本的PCR鉴定结果与分离培养对比,二者符合率为100％,模拟标本的检测灵敏度与纯菌结果一致。结论cytK基因为靶基因的PCR用于眼源性蜡样芽胞杆菌的快速检测,具有简便、快速、敏感、特异等特点,为眼内炎患者的快速诊断提供依据,在实际检验工作中有良好的应用前景。     

Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis

Bacillus anthracis has four plasmid possible virulence genotypes: pXO1+/pXO2+, pXO1+/pXO2-, pXO1-/pXO2+ or pXO1-/pXO2-. Due to the lack of a specific chromosomal marker for B. anthracis, differentiation of the pXO1-/pXO2- form of B. anthracis from closely related Bacillus cereus group species is difficult. In this study, we evaluate the ability of sspE, pXO1 and pXO2 primers to discriminate individual B. anthracis and the B. cereus group genotypes using multiplex real-time PCR and melting curve analysis. Optimal conditions for successful multiplex assays have been established. Purified DNAs from 38 bacterial strains including 11 strains of B. anthracis and 18 B. cereus group strains were analyzed. Nine of the B. cereus group near-neighbor strains were shown by multilocus sequence typing to be phylogenetically proximate to the B. anthracis clade. We have demonstrated that the four plasmid genotypes of B. anthracis and B. cereus group near-neighbors were differentially and simultaneously discriminated by this assay.     

Identity of hemolysins produced by Bacillus thuringiensis and Bacillus cereus

A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning.     

Identification of emetic toxin producing Bacillus cereus strains by a novel molecular assay

Bacillus cereus causes two types of gastrointestinal diseases: emesis and diarrhea. The emetic type of the disease is attributed to the heat-stable depsipeptide cereulide and symptoms resemble Staphylococcus aureus intoxication, but there is no rapid method available to detect B. cereus strains causing this type of disease. In this study, a polymerase chain reaction (PCR) fragment of unknown function was identified, which was shown to be specific for emetic toxin producing strains of B. cereus. The sequence of this amplicon was determined and a PCR assay was developed on this basis. One hundred B. cereus isolates obtained from different food poisoning outbreaks and diverse food sources from various geographical locations and 29 strains from other species belonging to the B. cereus group were tested by this assay. In addition, 49 non-B. cereus group strains, with special emphasis on food pathogens, were used to show that the assay is specific for emetic toxin producing B. cereus strains. The presented PCR assay is the first molecular tool for the rapid detection of emetic toxin producing B. cereus strains.     

Studies on the ATPase of Bacillus cereus.

The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified. The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation. The optimum temperature was 39.5 degrees C and the pH optimum at 7.5. On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K. The effect of various compounds on the enzymatic activity was studied. The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.     

Plasmid transformation of Bacillus cereus on cellophane membranes

A simple approach to test the ability of bacteria to undergo natural genetic transformation is suggested. The basic feature of the approach is the cultivation of bacterial cells in the presence of exogenous (plasmid) DNA on cellophane membranes placed successively on nutrient and selective agar. Using this approach the ability of Bacillus cereus for "natural" genetic transformation was detected. Transformation frequencies varied from 10(-8) to 10(-6).     

蜡样芽胞杆菌B905 hblA基因的初步分析

采用血平板培养的方法对蜡样芽胞杆菌905菌株溶血素BL检测,并通过PCR方法克隆其基因,结果表明该菌株产生溶血环且含有hblA、hblC、hblD溶血素BL全部基因;采用同源重组法构建了该菌株hblA基因缺失突变体,结果该菌株的溶血活性并未发生改变,可能是由于该菌株溶血素基因的结构与Handelsman构建所用的菌株Bacillus cereus UW85有一定的差异,或者是由于突变位点在阅读框内后端,未能真正破坏其表达。还需要进一步对其进行研究。