title: An electrochemical RNA hybridization assay for detection of the fecal indicator bacterium Escherichia coli.
abstract: Monitoring waters for indicator bacteria is required to protect the public from exposure to fecal pollution. Our proof-of-concept study describes a method for detecting fecal coliforms. The coliform Escherichia coli was used as a model fecal indicator. DNA probe-coated magnetic beads in combination with the electrochemical monitoring of the oxidation state of guanine nucleotides should allow for direct detection of bacterial RNA. To demonstrate this concept, we used voltammetry in connection with pencil electrodes to detect isolated E. coli 16S rRNA. Using this approach, 10(7) cells of E. coli were detected in a quantitative, reproducible fashion in 4h. Detection was achieved without a nucleic acid amplification step. The specificity of the assay for coliforms was demonstrated by testing against a panel of bacterial RNA. We also show that E. coli RNA can be detected directly from cell extracts. The method could be used for on-site detection and shows promise for adaptation into automated biosensors for water-quality monitoring.
authors.affiliation: Cooperative Institute for Marine and Atmospheric Studies, Rosenstiel School for Marine and Atmospheric Science, University of Miami, Miami, FL 33149, USA. michael.lagier@noaa.gov
authors.forename: Michael J
authors.initials: MJ
authors.lastname: LaGier
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Scholin
authors.forename: Jack W
authors.initials: JW
authors.lastname: Fell
authors.forename: Joseph
authors.initials: J
authors.lastname: Wang
authors.forename: Kelly D
authors.initials: KD
authors.lastname: Goodwin
mesh_terms: D001681:Biological Assay / Q000379:methods; D004563:Electrochemistry / Q000379:methods*; D004784:Environmental Monitoring / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005243:Feces / Q000382:microbiology*; D006150:Guanine Nucleotides / Q000378:metabolism; D008863:Microspheres; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D006150:Guanine Nucleotides; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S