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EHECO157:H7二重PCR的建立及应用

DOI: 10.7668/hbnxb.2011.06.012, PP. 58-66

Keywords: O157:H7,二重PCR,方法建立,细菌分离,样品检测

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Abstract:

建立用于从临床样品中检测EHECO157:H7的二重PCR方法。根据GenBank登录EHECO157:H7序列,通过对菌体和鞭毛抗原保守性核苷酸序列的比对和分析,分别设计编码O抗原rfbE基因和编码H抗原fliC基因各1对特异性引物,建立二重PCR检测方法,并以EHECO157:H7纯培养和模拟制备的3种阳性样品为原材料,对方法的敏感性和特异性进行分析。运用成功建立的二重PCR,从带菌率最高的牛粪便中检测EHECO157:H7,并进行菌株分离和鉴定。成功建立了rfbE与fliC的二重PCR方法,rfbE与fliC引物比例为2.5∶1,PCR循环参数中退火温度为56℃,扩增片段长度rfbE为812bp,fliC为625bp。检测阳性临床样品检测敏感性可以达到10cfu/g。检测24株非EHECO157:H7大肠杆菌和15株非大肠杆菌,只有EHECO157:H7能够扩增出特异性的rfbE条带,EHECO157:H7和EPECO55:H7能够扩增出fliC。只有EHECO157:H7能够同时扩增出rfbE与fliC2条目的条带。从63份牛粪便样品中分离到4株EHECO157:H7:生化试验表明4株EHECO157:H7具有典型EHECO157:H7的生化特性;药敏试验表明4株EHECO157:H7具有较为广谱的耐药性;rfbE序列分析与GenBank序列同源性介于97.3%~99.2%之间,fliC序列与GenBank序列同源性介于97.6%~100%之间;4株EHECO157:H7对Balb/c小鼠的致病性有差异,EHECO157:H728#和EHECO157:H738#致病性强,EHECO157:H73#和EHECO157:H717#较弱。以rfbE和fliC为目的基因成功建立了二重PCR,敏感性和特异性良好,可用于临床样品的检测,提高细菌分离率。

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