Optimization of a 3'-minor groove binder-DNA probe targeting the uidA gene for rapid identification of Escherichia coli O157:H7 using real-time PCR.
Enterohemorrhagic Escherichia coli are harmful human pathogens capable of causing bloody diarrhea and vomiting. An important serotype commonly associated with human illness is the E. coli O157:H7 serotype. Unlike other real-time polymerase chain reaction (PCR) methods for identifying E. coli O157:H7, this study describes the development and optimization of a real-time PCR method targeting a conserved point mutation at +93 in the uidA (gusA) gene that is unique to O157:H7, distinguishing it from non-O157:H7 serotypes. A TET-labeled Minor Groove Binder (MGB) DNA probe was designed for use in a 5' nuclease PCR assay. Using a panel of two E. coli O157:H7 strains, three E. coli non-O157:H7 strains, and one non-E. coli species, the assay was optimized for the specific detection of the E. coli O157:H7 strains. Optimal conditions were identified at high anneal/extend temperatures, low magnesium concentrations, and low probe concentrations, resulting in correct identification of E. coli O157:H7 and non-O157:H7 strains. The improved specificity of MGB probes for single base pair mismatches such as the +93 uidA mutation provides a novel approach towards rapid identification of E. coli O157:H7.
Yoshitomi
Ken J
KJ
Seafood Products Research Center, US Food and Drug Administration, 22201 23rd Drive SE, Bothell, WA 98021-4421, USA. ken.yoshitomi@fda.gov
Jinneman
Karen C
KC
Weagant
Stephen D
SD
D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D019453:Escherichia coli O157 / Q000235:genetics*; D006801:Humans; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial