《微生物之棒状杆菌.ppt》由会员分享，可在线阅读，更多相关《微生物之棒状杆菌.ppt（23页珍藏版）》请在三一办公上搜索。
1、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
2、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
3、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
4、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
5、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.dip
6、htheriae）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
7、,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.Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by
8、 PCRJ.Veterinary Microbiology 88(2002)7583.2 Fabiola Mancini,Monica Monaco,Marco Pataracchia,et al.Identification and molecular discrimination of toxigenic and nontoxigenic diphtheria Corynebacterium strains by combined real-time polymerase chain reaction assaysJ.Diagnostic Microbiology and Infectio
9、us Disease 73(2012)111120.3 Fabricia P.Pimenta,Gisele A.M.Matias,Gabriela A.Pereira,et al.A PCR for dtxR gene:Application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriaeJ.Molecular and Cellular Probes 22(2008)189192.4 Luis G.C.Pacheco,Roberta R.Pena,Thiago L.P.Castro,et al.Mu
10、ltiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samplesJ.Journal of Medical Microbiology(2007),56,480486.5 Ketevan Kobaidze,Tanja Popovic,1Hiroshi Nakao,et al.Direct Polymerase Chain Reaction for Detec
11、tion of Toxigenic Corynebacterium diphtheriae Strains from the Republic of Georgia after Prolonged StorageJ.The Journal of Infectious Diseases 2000;181(Suppl 1):S1525.,6 M J Pallen,A J Hay,L H Puckey,et al.Polymerase chain reaction for screening clinical isolates of corynebacteria for the production
12、 of diphtheria toxinJ.Clin Pathol 1994;47:353-356.7 DOMINIQUE AUBEL,FRANCOIS N.R.RENAUD,AND JEAN FRENEY,et al.Genomic Diversity of Several Corynebacterium Species Identified by Amplification of the 16s-23s rRNA Gene Spacer RegionsJ.INTERNATIONJOAULR NAL OF SYSTEMATBIACC TERIOLOJGuYly,1997,p.767-772.
13、8 Andreas Sing,Anja Berger,Wulf Schneider-Brachert,et al.Rapid Detection and Molecular Differentiation of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans Strains by LightCycler PCRJ.JOURNAL OF CLINICAL MICROBIOLOGY,July 2011,p.24852489.9 Pamela K.Cassiday,Lucia C.Pawloski,Tejprata
14、p Tiwari,et al.Analysis of Toxigenic Corynebacterium ulcerans Strains Revealing Potential for False-Negative Real-Time PCR ResultsJ.JOURNAL OF CLINICAL MICROBIOLOGY,Jan.2008,p.331333.10 Takako Komiya,Yukiji Seto,Aruni De Zoysa,et al.Two Japanese Corynebacterium ulcerans isolates from the same hospit
15、al:ribotype,toxigenicity and serum antitoxin titreJ.Journal of Medical Microbiology(2010),59,14971504.11 Aron J.Hall,Pamela K.Cassiday,Kathryn A.Bernard,et al.Novel Corynebacterium diphtheriae in Domestic CatsJ.Emerging Infectious Diseases Vol.16,No.4,April 2010.,12 Fabricia P.Pimenta,Raphael Hirata
16、 Jr,Ana C.P.Rosa,et al.A multiplex PCR assay for simultaneous detection of Corynebacterium diphtheriae and differentiation between non-toxigenic and toxigenic isolatesJ13 Regina Schuhegger,Marion Lindermayer,Rudolf Kugler.Detection of Toxigenic Corynebacterium diphtheriae and Corynebacterium ulceran
17、s Strains by a Novel Real-Time PCRJ.JOURNAL OF CLINICAL MICROBIOLOGY,Aug.2008,p.28222823.14 Elizabeth A.Mothershed,Pamela K.Cassiday,et al.Development of a Real-Time Fluorescence PCR Assay for Rapid Detection of the Diphtheria Toxin GeneJ.JOURNAL OF CLINICAL MICROBIOLOGY,Dec.2002,p.47134719.15 M J P
18、allen,A J Hay,L H Puckey,et al.Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxinJ.J Clin Pathol 1994;47:353-356.16 HIROSHI NAKAO AND TANJA POPOVIC.Development of a Direct PCR Assay for Detection of the Diphtheria Toxin GeneJ.JOURNAL OF CLINICAL MICROBIOLOGY,July 1997,p.16511655,
