Use of electrochemical DNA biosensors for rapid molecular identification of uropathogens in clinical urine specimens.
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.
Liao
Joseph C
JC
Division of Infectious Diseases, 111F, VA Greater LA Healthcare, Los Angeles, CA 90073, USA.
Mastali
Mitra
M
Gau
Vincent
V
Suchard
Marc A
MA
Møller
Annette K
AK
Bruckner
David A
DA
Babbitt
Jane T
JT
Li
Yang
Y
Gornbein
Jeffrey
J
Landaw
Elliot M
EM
McCabe
Edward R B
ER
Churchill
Bernard M
BM
Haake
David A
DA
D015374:Biosensing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis*; D004563:Electrochemistry / Q000379:methods*; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology; D006801:Humans; D013045:Species Specificity; D014552:Urinary Tract Infections / Q000175:diagnosis / Q000382:microbiology*; D014556:Urine / Q000382:microbiology*
D004269:DNA, Bacterial