Detection of Escherichia coli O157 using equal-length double-stranded fluorescence probe in a real-time polymerase chain reaction assay.
BACKGROUND
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
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.
Guangfu
G
Yu
Jianjun
J
Niu
Mingshan
M
Shen
Hanjuan
H
Shao
Liang
L
Chen
D004247:DNA / Q000737:chemistry / Q000235:genetics; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000821:virology*; D019453:Escherichia coli O157 / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D004247:DNA