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

• Published in: Molecular and cellular probes 2003-12, Vol.17 (6), p.275-280
• Main Authors: Yoshitomi, Ken J, Jinneman, Karen C, Weagant, Stephen D
• Format: Article
• Language: English
• Subjects: DNA Primers - genetics; DNA Probes - chemistry; DNA Probes - genetics; DNA, Bacterial - genetics; Escherichia coli O157 - genetics; Escherichia coli O157:H7; Humans; Minor groove binder probe; Point Mutation - genetics; Polymerase Chain Reaction; Sensitivity and Specificity; uidA
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2003.07.001

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.