Development of a multiplex real-time polymerase chain reaction for simultaneous detection of enterohemorrhagic Escherichia coli and enteropathogenic Escherichia coli strains.
A multiplex real-time polymerase chain reaction (PCR) was developed for the simultaneous detection of genes encoding intimin (eae) and all variants of Shiga toxins 1 and 2 (stx1 and stx2) in diagnostic samples. The uidA gene encoding a beta-glucuronidase specific for Escherichia coli and Shigella spp. was included in the multiplex PCR assay as an internal amplification control. The multiplex PCR was tested on 30 E. coli reference strains and 174 diagnostic samples already characterized as harboring stx1, stx2, and eae genes. The multiplex PCR correctly detected the genes in all strains examined. No cross reaction was observed with 68 strains representing other gastrointestinal pathogens, normal gastrointestinal flora, or closely related bacteria, reflecting 100% specificity of the assay. The detection limits of the multiplex PCR were 5 genome equivalents for stx2 and 50 genome equivalents for eae and stx1.
Bavarian Health and Food Safety Authority, Oberschleissheim, Germany.
Melanie
M
Pavlovic
Ingrid
I
Huber
Henrike
H
Skala
Regina
R
Konrad
Herbert
H
Schmidt
Andreas
A
Sing
Ulrich
U
Busch
D018829:Adhesins, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D054324:Enterohemorrhagic Escherichia coli / Q000302:isolation & purification*; D054308:Enteropathogenic Escherichia coli / Q000302:isolation & purification*; D004926:Escherichia coli / Q000145:classification / Q000302:isolation & purification / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000175:diagnosis*; D029968:Escherichia coli Proteins / Q000235:genetics; D005243:Feces / Q000382:microbiology; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1 / Q000235:genetics; D022641:Shiga Toxin 2 / Q000235:genetics; D014774:Virulence / Q000235:genetics
D018829:Adhesins, Bacterial; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C073819:eaeA protein, E coli