Journal
MICROCHIMICA ACTA
Volume 180, Issue 13-14, Pages 1233-1240Publisher
SPRINGER WIEN
DOI: 10.1007/s00604-013-1061-0
Keywords
Electrochemical DNA sensor; Enteropathogenic Escherichia coli; bfpA gene; PCR
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Funding
- National Natural Science Foundation of China [21075141, 81101638]
- Special Fund Project of Chongqing Key Laboratory (CSTC)
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We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 10(7) CFU mL(-1) levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.
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