EHEC O157：H7二重PCR的建立及应用

Duplex PCR Procedure for the Detection of EHEC O157:H7

建立用于从临床样品中检测EHEC O157:H7的二重PCR方法。根据GenBank登录EHEC O157: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EHEC O157: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EHEC O157: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812 bp,fliC为625 bp。检测阳性临床样品检测敏感性可以达到10 cfu/g。检测24株非EHEC O157: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,EHEC O157:H7和EPEC O55:H7能够扩增出fliC。只有EHEC O157:H7能够同时扩增出rfbE与fliC 2条目的条带。从63份牛粪便样品中分离到4株EHEC O157:H7:生化试验表明4株EHEC O157:H7具有典型EHECO157:H7的生化特性;药敏试验表明4株EHEC O157: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EHEC O157:H7对Balb/c小鼠的致病性有差异,EHEC O157:H728#和EHEC O157:H738#致病性强,EHEC O157:H73#和EHEC O157: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。

Construct a duplex PCR for the detection of E.coli O157：H7 from clinical samples.Analyses of the available sequences of the two major virulence genes listed in GenBank allowed us to develop the two-gene multiplex PCR protocol that maintained the specificity of each primer pair.Sensitivity and specificity were verified by detection of E.coli O157：H7 culture and prepared E.coli O157：H7 positive samples.Applying the duplex PCR,we detect E.coli O157：H7from bovine feces and isolate the positive samples.The resulting two bands for rfbE and fliC were even and distinct with product sizes of 812 and 625 bp,respectively.The proportion of rfbE and fliC of PCR amplification reaction was 2.5 ∶ 1（V/V）and the optimal PCR amplification temperature was 56℃.Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104 cfu/g.After a 6-h enrichment of E.coli O157-spiked samples,a sensitivity level of 10 cfu/g was achieved.The procedure was validated with a total of 24 E.coli strains as well as 15 non-E.coli strains.Specificity of the O antigen was indicated by amplification of only E.coli O157,and not other E.coli serotypes and non-E.coli strains.Specificity of the H antigen was indicated by amplification of only E.coli O157 and E.coli O55,and not others.From 63 bovine feces,we obtained 4 isolates of E.coli O157：H7.Biochemical feature tests showed that isolated E.coli O157：H7 have the representative feature of E.coli O157：H7.Susceptibility tests indicated that the four E.coli O157：H7 have broad-spectrum drug tolerance.Sequence analyses of rfbE and fliC suggested that the four isolates have homology of 97.3%-100%.The four E.coli O157：H7 isolates showed different pathogenicity,isolate 28# and 38# were high,3# and 17# low.We successfully developed the duplex PCR for O157：H7 for the detection of clinical samples.