Identification of Escherichia coli O157:H7 and other enterohemorrhagic serotypes by EHEC- hlyA targeting, strand displacement amplification, and fluorescence polarization.
Human disease caused by enterohemorrhagic E. coli O157:H7 and other serotypes (EHEC) has been associated with bovine fecal contamination of food and the environment. The range of serotypes, low infectious dose, and numerous transmission vehicles for EHEC render development of detection methods for this pathogen complex. In this study, the hemolysin gene (EHEC- hly A) was targeted with oligonucleotides, and probe-target hybrids were amplified using strand displacement amplification (SDA). Amplicons were resolved in the complete reaction mix through changes in the fluorescence polarization (FP) of a fluorescein-labeled detector probe hybridized to the amplicons during amplification. Results combining EHEC- hly A, SDA, and FP were obtained within 35 min of reaction initiation. The test specificity was determined on EHEC strains representing 13 serotypes (49 isolates); and control uropathogenic, commensal, and other organisms (10 isolates). Statistical analysis of results indicated a sensitivity in the reaction vessel to 4.3 bacteria (95% confidence interval), and a specificity for EHEC (n=59) at 100% (P=5.11E-17; i.e. P<<0.05). Detection based on combining EHEC- hly A, SDA, and FP was compatible with water sources directly associated with human infection (drinking and recreational supplies), and bovine drinking trough water representing an environmental matrix linked to the maintenance of an EHEC animal reservoir.
Ge
B
B
Department of Nutrition and Food Science, University of Maryland, College Park, Maryland, MD 20742, USA.
Larkin
C
C
Ahn
S
S
Jolley
M
M
Nasir
M
M
Meng
J
J
Hall
R H
RH
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D001431:Bacteriological Techniques; D002417:Cattle; D003603:Cytotoxins / Q000235:genetics; D019453:Escherichia coli O157 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005454:Fluorescence Polarization; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D012680:Sensitivity and Specificity; D012703:Serotyping; D014871:Water Microbiology
D001427:Bacterial Toxins; D003603:Cytotoxins; C097321:EHEC-hlyA protein, E coli; D029968:Escherichia coli Proteins