Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection.
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.
Bush
C E
CE
Department of Molecular Diagnostics, Baxter Diagnostics Inc., West Sacramento, California 95691.
Vanden Brink
K M
KM
Sherman
D G
DG
Peterson
W R
WR
Beninsig
L A
LA
Godsey
J H
JH
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
D005456:Fluorescent Dyes; D008674:Metals, Rare Earth; D015345:Oligonucleotide Probes; D011137:Polystyrenes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal