Electrocatalytic detection of pathogenic DNA sequences and antibiotic resistance markers.
The detection of specific DNA sequences using electrochemical readout would permit the rapid and inexpensive detection and identification of bacterial pathogens. A new assay developed for this purpose is described that harnesses a sensitive electrocatalytic process to monitor DNA hybridization. Two sequences belonging to the pathogenic microbe Helicobacter pylori are used to demonstrate the versatility and specificity of the assay: one that codes for an unique H. pylori protein and one that represents a small portion of the 23S rRNA from this organism. Both sequences can be monitored into the nanomolar concentration range. Target sequences introduced to the electrode surface as synthetic oligonucleotides, PCR products, and RNA transcripts are all detected with high specificity. In addition to reporting the presence of pathogen-related sequences, this assay can accurately resolve single-base changes in target sequences. An A2143C substitution within the H. pylori rRNA that confers antibiotic resistance significantly attenuates hybridization to an immobilized probe corresponding to the WT sequence. The single-base mismatch introduced by this mutation slows the kinetics of hybridization and permits discrimination of the two sequences when short hybridization times are employed. The remarkable sensitivity of this label-free assay to small sequence changes may provide the basis of a new method for the detection and genotyping of infectious bacteria using electrochemical methods.
Lapierre
Melissa A
MA
Eugene F Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
O'Keefe
Meaghan
M
Taft
Bradford J
BJ
Kelley
Shana O
SO
D001426:Bacterial Proteins / Q000235:genetics; D020137:Base Pair Mismatch / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics*; D004563:Electrochemistry; D005819:Genetic Markers / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification*; D007501:Iron / Q000737:chemistry; D010084:Oxidation-Reduction; D017354:Point Mutation / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D012428:Ruthenium / Q000737:chemistry; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D015342:DNA Probes; D004269:DNA, Bacterial; D005819:Genetic Markers; D012338:RNA, Ribosomal, 23S; D012428:Ruthenium; D007501:Iron