title: Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection.
abstract: The development of technology to increase the sensitivity and speed of detection of bacterial pathogens in samples is important for diagnosis and monitoring of illness. We have developed a sensitive and rapid method for the detection of bacteria, using Escherichia coli as a model, which combines transcription-based target amplification with a bead-based sandwich hybridization assay using rare earth metal chelate labelled probes and time-resolved fluorescence detection. Using these methods as little as 100 copies (0.00016 attomoles) of purified native Escherichia coli rRNA or just one bacterial cell in a spiked sample could be detected. These results demonstrate that amplification of rRNA by transcription-based amplification and detection by time-resolved fluorescence provide a sensitive technology for the direct detection of micro-organisms without the requirement for prior cultivation.
authors.affiliation: Department of Molecular Diagnostics, Baxter Diagnostics Inc., West Sacramento, California 95691.
authors.forename: C E
authors.initials: CE
authors.lastname: Bush
authors.forename: K M
authors.initials: KM
authors.lastname: Vanden Brink
authors.forename: D G
authors.initials: DG
authors.lastname: Sherman
authors.forename: W R
authors.initials: WR
authors.lastname: Peterson
authors.forename: L A
authors.initials: LA
authors.lastname: Beninsig
authors.forename: J H
authors.initials: JH
authors.lastname: Godsey
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D006801:Humans; D008674:Metals, Rare Earth; D008863:Microspheres; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes; D011137:Polystyrenes; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D008674:Metals, Rare Earth; D015345:Oligonucleotide Probes; D011137:Polystyrenes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal