Real-time nucleic acid sequence-based amplification (NASBA) using an adenine-induced quenching probe and an intercalator dye.
OBJECTIVE
We found that an adenine base caused fluorescence quenching of a fluorescein (FL)-labelled probe in DNA:RNA hybrid sequences, and applied this finding to a nucleic acid sequence-based amplification (NASBA) method.


RESULTS
The present NASBA method employed a probe containing an FL-modified thymine at its 3' end and ethidium bromide (EtBr) on the basis of a combination of adenine-induced quenching and fluorescence resonance energy transfer (FRET) between the FL donor and EtBr acceptor. This NASBA was used to detect Shiga toxin (STX) stx-specific mRNA in STX-producing Escherichia coli, demonstrating rapid quantification of the target gene with high sensitivity.


CONCLUSIONS
Although the inherent quenching effect of adenine was inferior to that of guanine, FRET between the FL and EtBr moieties enhanced the adenine-induced quenching, allowing rapid and sensitive real-time NASBA detection.


CONCLUSIONS
This study gives a novel real-time diagnostic system based on NASBA for a sensitive mRNA (or viral RNA) detection.
Kouguchi
Y
Y
Fukuyama City Public Health Center, Fukuyama, Hiroshima, Japan. shiken-kensa@city.fukuyama.hiroshima.jp
Teramoto
M
M
Kuramoto
M
M
D000225:Adenine / Q000737:chemistry / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D007364:Intercalating Agents / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000378:metabolism; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D022621:Shiga Toxin / Q000235:genetics; D054323:Shiga-Toxigenic Escherichia coli / Q000235:genetics
D004269:DNA, Bacterial; D007364:Intercalating Agents; D012329:RNA, Bacterial; D022621:Shiga Toxin; D000225:Adenine