微生物之棒状杆菌.ppt

棒状杆菌属种属及毒力鉴定,江苏嘉语生物医药技术有限公司 陈颖曦,内容概要,棒状杆菌属及白喉棒状杆菌简介,棒状杆菌属(Corynebacterium)是一群革兰染色阳性杆菌。本属细菌的特点是菌体一端或两端膨大呈棒状，菌体染色不均匀，可出现异染颗粒。该菌属无荚膜、无鞭毛，不产生芽胞。种类较多，广泛分布于动、植物。可在人体皮肤、上呼吸道和泌尿生殖道粘膜等处定居。大多为非致病菌或条件致病菌，其中白喉棒状杆菌是唯一能引起人类致病且具较强传染性的菌种。,白喉棒状杆菌的异染颗粒 （Albert染色 ）,白喉棒状杆菌(C.diphtheriae)，俗称白喉杆菌，是白喉的病原菌。白喉是一种急性呼吸道传染病，患者咽喉部粘膜可出现灰白色的假膜。该菌能产生强烈外毒素，进入血液可引起全身中毒症状。菌体细长微弯，一端或两端膨大呈棒状。细菌排列不规则，常排列呈V、L等形状。革兰染色阳性。用美蓝短时间染色菌体着色不均匀，出现有深染的颗粒。用Neisser或Albert 等法染色，这些颗粒与菌体着染颜色不同，称为异染颗。颗粒的主要成分是核糖核酸和多偏磷酸盐，在鉴定时有重要意义。但当细菌衰老时异染颗粒被消耗而不明显，且细胞壁变薄易被脱色，常造成革兰染色不定，有时可表现为在革兰阴性的菌体中见有阳性颗粒或节段。 需氧或兼性厌氧。在血平板上可生长，菌落大小13mm。在含有凝固血清的吕氏血清斜面上生长迅速，涂片染色异染颗粒明显。分离培养时常用亚碲酸钾血平板作为鉴别选择培养基。因亚碲酸钾能抑制杂菌，而白喉棒状杆菌不受影响，并能吸收亚碲酸盐使其还原为黑色的金属碲，菌落呈黑色。白喉棒状杆菌在亚碲酸钾血平板上可形成三种不同形态特征的菌落，分别称为重型、轻型和中间型。三型的产毒株与疾病的轻重程度无明显的对应关系，但对流行病学分析具有一定意义。在我国以轻型多见。 白喉棒状杆菌对湿热抵抗力不强，58C 10分钟或煮沸1分钟死亡。但对寒冷、干燥和日光的抵抗力较其他无芽胞菌强。在衣服、床单、玩具上可存活数天至数周。对常用消毒剂抵抗力较弱，5%石碳酸1分钟、3%来苏尔10分钟处理可杀灭。对青霉素、氯霉素、红霉素等敏感。,棒状杆菌与疾病,常用种属鉴定靶点及序列比对,种属鉴定靶点的选定,16S作为国际公认的细菌分类“金标准”，作为细菌属的鉴定靶点是得到认可的，在已下载到的棒状杆菌文献中，缺乏关于棒状杆菌属鉴定靶点的描述，介于16S在分类上应用的普遍性和权威性，在此将16S暂定为棒状杆菌属的鉴定靶点。 代谢调控因子编码基因dtxR和RNA聚合酶亚基B编码基因rpoB是白喉棒状杆菌鉴定讨论最多的靶点，特别是dtxR在白喉的鉴定中表现出良好的特异性，现将dtxR作为白喉的首选鉴定靶点，rpoB作为备选，以区分白喉和非白喉。换句话说，只要16S显阳性，排除假阳性的结果，就认为是棒状杆菌属的细菌，再加上dtxR和rpoB显阳性，排除假阳性结果，就说明是白喉，如果是阴性就说明是棒状杆菌的其他种，其他种在这不进行细致的鉴定。,临床上棒状杆菌中的致病种包括白喉棒状杆菌在内，有36个种，然而在此不可能将数目众多的种都考虑进去，产毒白喉棒状杆菌（ Cdiphtheriae ）是最主要的致病种，也是绝对致病种，在本课题中作为重点考虑，此外，在16S的比对中将条件致病种Cpseudodiphtheriticum， Culcerans，Camycolatum，Cstriatum，Cjeikeium， Curealyticum和Cxerosis考虑进去，另加跟牛乳腺炎有关的2个种：Cbovis和Cpyogenes。,16S序列比对,选取白喉棒状杆菌（C.diphtheriae ）等10个种的16S序列进行比对，共计278条序列。,棒状杆菌属鉴定16S-1,由于文献都是直接用靶基因鉴定到种，没有用16S属的鉴定棒状杆菌属的引物，属引物还需自行设计。,dtxR序列比对,选取白喉棒状杆菌21条dtxR序列进行比对。,文献中dtxR引物,rpoB序列比对,选取白喉（ Cdiphtheriae ）等9个种的rpoB序列进行比对，合计102条。,文献中的棒状杆菌rpoB引物,棒状杆菌毒力因子及序列比对,白喉毒素tox基因比对,选取13株C.diphtheriae的白喉tox基因和15株C.ulcerans的tox基因进行比对。,文献中白喉毒素tox引物,白喉毒素又A,B2个亚基组成，但某些菌株只存在一种亚基，另一亚基丢失的情况，建议针对这两个亚基分别设计1对引物检测，方能对菌株是否含有白喉毒素进行准确定性。,鞘磷脂酶PLD基因比对,选取10株C.ulcerans的PLD基因进行比对,选取17株C.pseudotuberculosis的PLD基因进行比对,文献中PLD引物,参考文献,1 Burhan Cetinkayaa,*, Murat Karahana, Eray Atila,et al. 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