Rapid, species-specific detection of uropathogen 16S rDNA and rRNA at ambient temperature by dot-blot hybridization and an electrochemical sensor array.
Development of rapid molecular approaches for pathogen detection is key to improving treatment of infectious diseases. For this study, the kinetics and temperature-dependence of DNA probe hybridization to uropathogen species-specific sequences were examined. A set of oligonucleotide probes were designed based on variable regions of the 16S gene of the Escherichia coli, Proteus mirabilis, Klebsiella oxytoca, and Pseudomonas aeruginosa. A universal bacterial probe and probes-specific for gram-positive and gram-negative organisms were also included. The oligonucleotide probes discriminated among 16S genes derived from 11 different species of uropathogenic bacteria applied to nylon membranes in a dot-blot format. Significant binding of oligonucleotide probes to target DNA and removal of nonspecific binding by membrane washing could both be achieved rapidly, requiring as little as 10 min. An oligonucleotide probe from the same species-specific region of the E. coli 16S gene was used as a capture probe in a novel electrochemical 16-sensor array based on microfabrication technology. Sequence-specific hybridization of target uropathogen 16S rDNA was detected through horseradish peroxidase acting as an electrochemical transducer via a second, detector probe. The sensor array demonstrated rapid, species-specific hybridization in a time course consistent with the rapid kinetics of the dot-blot hybridization studies. As in the dot-blot hybridization studies, species-specific detection of bacterial nucleic acids using the sensor array approach was demonstrated both at 65 degrees C and at room temperature. These results demonstrate that molecular hybridization approaches can be adapted to rapid, room temperature conditions ideal for an electrochemical sensor array platform.
Sun
Chien-Pin
CP
Biomedical Engineering Department, Henry Samueli School of Engineering and Applied Science, UCLA, Los Angeles, CA, USA.
Liao
Joseph C
JC
Zhang
Yao-Hua
YH
Gau
Vincent
V
Mastali
Mitra
M
Babbitt
Jane T
JT
Grundfest
Warren S
WS
Churchill
Bernard M
BM
McCabe
Edward R B
ER
Haake
David A
DA
D001419:Bacteria / Q000235:genetics*; D001483:Base Sequence; D003141:Communicable Diseases / Q000175:diagnosis* / Q000382:microbiology; D004563:Electrochemistry; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012588:Scintillation Counting; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S