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Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

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Abstract

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 107 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.

An electrochemical DNA sensor was first designed to detect a bfpA gene specifically related to the EPEC.

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Acknowledgment

This work was funded by the National Natural Science Foundation of China (21075141 and 81101638) and supported by the Special Fund Project of Chongqing Key Laboratory (CSTC).

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Authors and Affiliations

  1. Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Wei Zhang, Caihui Luo, Liang Zhong, Dan Zhao & Shijia Ding

  2. Molecular Oncology and Epigenetics Laboratory, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People’s Republic of China

    Wei Cheng

  3. Detachment of Criminal Police, Yongchuan District Public Security Bureau, Chongqing, 400016, People’s Republic of China

    Shichang Nie

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  1. Wei Zhang
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  2. Caihui Luo
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Correspondence to Shijia Ding.

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Zhang, W., Luo, C., Zhong, L. et al. Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor. Microchim Acta 180, 1233–1240 (2013). https://doi.org/10.1007/s00604-013-1061-0

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  • DOI: https://doi.org/10.1007/s00604-013-1061-0

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