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Journal of Clinical Microbiology, February 2006, p. 561-570, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.561-570.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Use of Electrochemical DNA Biosensors for Rapid Molecular Identification of Uropathogens in Clinical Urine Specimens

Departments of Urology,¹ Medicine,² Biomathematics,⁴ Human Genetics,⁵ Pathology and Laboratory Medicine,⁶ Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, California 90095,⁷ GeneFluidics Inc., Monterey Park, California 91754,³ Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073⁸

Received 28 July 2005/ Returned for modification 28 October 2005/ Accepted 13 November 2005

We describe the first species-specific detection of bacterial pathogens in human clinical fluid samples using a microfabricated electrochemical sensor array. Each of the 16 sensors in the array consisted of three single-layer gold electrodes—working, reference, and auxiliary. Each of the working electrodes contained one representative from a library of capture probes, each specific for a clinically relevant bacterial urinary pathogen. The library included probes for Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Enterocococcus spp., and the Klebsiella-Enterobacter group. A bacterial 16S rRNA target derived from single-step bacterial lysis was hybridized both to the biotin-modified capture probe on the sensor surface and to a second, fluorescein-modified detector probe. Detection of the target-probe hybrids was achieved through binding of a horseradish peroxidase (HRP)-conjugated anti-fluorescein antibody to the detector probe. Amperometric measurement of the catalyzed HRP reaction was obtained at a fixed potential of –200 mV between the working and reference electrodes. Species-specific detection of as few as 2,600 uropathogenic bacteria in culture, inoculated urine, and clinical urine samples was achieved within 45 min from the beginning of sample processing. In a feasibility study of this amperometric detection system using blinded clinical urine specimens, the sensor array had 100% sensitivity for direct detection of gram-negative bacteria without nucleic acid purification or amplification. Identification was demonstrated for 98% of gram-negative bacteria for which species-specific probes were available. When combined with a microfluidics-based sample preparation module, the integrated system could serve as a point-of-care device for rapid diagnosis of urinary tract infections.

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