Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection

• Published in: Molecular and cellular probes Vol. 5; no. 6; pp. 467 - 472
• Main Authors: Bush, Charlene E., Vanden Brink, Kurt M., Sherman, David G., Richard Peterson, W., Beninsig, Laura A., Godsey, James H.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 01.12.1991; Elsevier
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Base Sequence; Biological and medical sciences; Escherichia coli; Escherichia coli - isolation & purification; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Humans; Metals, Rare Earth; Microbiology; Microspheres; Molecular Sequence Data; Nucleic Acid Amplification Techniques; Oligonucleotide Probes; Polystyrenes; RNA, Bacterial - genetics; RNA, Bacterial - isolation & purification; RNA, Ribosomal - genetics; RNA, Ribosomal - isolation & purification; Sensitivity and Specificity; time-resolved fluorescence detection; transcription-based amplification; time-resolved fluorescence detection; Escherichia coli; transcription-based amplification; Time resolution; Gene amplification; Rapid technique; Ribosomal RNA; Pathogen strain; Fluorescence; Bacteria; Medical screening; Enterobacteriaceae
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/S0890-8508(05)80019-2

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.