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Real‐time nucleic acid sequence–based amplification (NASBA) using an adenine‐induced quenching probe and an intercalator dye
Author(s) -
Kouguchi Y.,
Teramoto M.,
Kuramoto M.
Publication year - 2010
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2010.04801.x
Subject(s) - nasba , intercalation (chemistry) , nucleic acid , quenching (fluorescence) , chemistry , nucleic acid detection , sequence (biology) , microbiology and biotechnology , biochemistry , nucleic acid sequence , biophysics , computational biology , biology , dna , fluorescence , organic chemistry , physics , quantum mechanics
Aims: 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. Methods and 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. Conclusion: 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. Significance and Impact of the Study: This study gives a novel real‐time diagnostic system based on NASBA for a sensitive mRNA (or viral RNA) detection.