Yu Guangfu, Niu Jianjun, Shen Mingshan, Shao Hanjuan, Chen Liang
The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, 422 Siming Nan Road, Xiamen 361005, Fujian, People's Republic of China.
Clin Chim Acta. 2006 Apr;366(1-2):281-6. doi: 10.1016/j.cca.2005.10.027. Epub 2006 Feb 14.
Enterohemorrhagic Escherichia coli (E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli (E. coli) O157.
A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen.
The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 x 10(3) CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative.
By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157.
肠出血性大肠杆菌O157是一种危险的病原体，可导致血性腹泻和严重的溶血尿毒综合征（HUS）。尽管已经整合了几种基于实时聚合酶链反应（PCR）的检测系统来检测这种病原体，但大多数系统并不特异。我们报告了一种针对rfbE基因的实时定量PCR方法，rfbE基因在大肠杆菌O157中特异性表达。因此，该方法可用于诊断肠出血性大肠杆菌O157。
开发了一种基于核酸的诊断检测系统，该系统结合了等长双链荧光探针技术和实时PCR来检测大肠杆菌O157。该检测系统利用高度保守的rfbE O抗原合成基因，在实时PCR中使用一对与rfbE基因互补的荧光淬灭探针来定量病原体的存在。
通过比较对14种不同相关病原体（包括普通大肠杆菌、侵袭性大肠杆菌（EIEC）、沙门氏菌、志贺氏菌和大肠杆菌O157）的检测结果，评估了诊断方法的特异性。使用大肠杆菌O157标准样品的10倍系列稀释液确定该方法的检测限，低至1.49×10³CFU/ml即可检测到。所有表达rfbE基因的O157血清型大肠杆菌在该检测中均为阳性，而所有其他不表达rfbE基因的物种均为阴性。
通过结合等长双链荧光探针技术和实时PCR，我们开发了一种简单、快速、特异且灵敏的方法来检测大肠杆菌O157。
