Real-time nucleic acid sequence-based amplification (NASBA) using an adenine-induced quenching probe and an intercalator dye

Journal of Applied Microbiology
Y KouguchiM Kuramoto

Abstract

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. 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. 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. This study gives a novel real-time diagnostic system based on NASBA for a sensitive mRNA (or viral RNA) detection.

References

Mar 7, 1991·Nature·J Compton
Oct 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·J C GuatelliT R Gingeras
Mar 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·J C GuatelliT R Gingeras
Aug 11, 1993·Nucleic Acids Research·L G LeeW Bloch
Jan 22, 1998·Applied and Environmental Microbiology·J ChenM Griffiths
Feb 3, 1998·Nature Biotechnology·S TyagiF R Kramer
Jan 27, 2000·Journal of Biochemical and Biophysical Methods·K NathC J Di Como
Sep 6, 2002·Genome Research·W Mathias HowellAnthony J Brookes
Jun 29, 2005·Clinical Biochemistry·Anikó SzilvásiAttila Tordai
Sep 1, 2005·Journal of Agricultural and Food Chemistry·Marta HernándezMaria Pla
Jan 16, 2007·Applied and Environmental Microbiology·Else M FykseJanet M Blatny
Jul 3, 2007·Analytical Biochemistry·Naoko MatsumotoSetsuo Hasegawa
Mar 11, 2008·Leukemia Research·Ruriko TanakaShinya Kimura

❮ Previous
Next ❯

Citations

Aug 7, 2021·Journal of Immunoassay & Immunochemistry·Mekbib AstatkeAmy Connolly

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.