Label-free fluorometric detection of influenza viral RNA by strand displacement coupled with rolling circle amplification

Since influenza occurs globally every year, it is important to develop a facile and accurate method to detect the influenza virus. This study aimed at developing a sensitive fluorometric assay for detecting influenza viral RNA through tandem gene amplification methods including reverse transcription...

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Bibliographic Details
Published in:Analyst (London) 2021-01, Vol.145 (24), p.82-87
Main Authors: Lee, Hyobeen, Kim, Dong-Min, Kim, Dong-Eun
Format: Article
Language:English
Subjects:
Amplification
Deoxyribonucleic acid
DNA
Fluorescence
Fluorometry
Gene amplification
Humans
Influenza
Influenza A Virus, H1N1 Subtype
Influenza A Virus, H3N2 Subtype
Influenza, Human - diagnosis
Nucleic Acid Amplification Techniques
Ribonucleic acid
RNA
RNA, Viral - genetics
Viruses
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Summary:Since influenza occurs globally every year, it is important to develop a facile and accurate method to detect the influenza virus. This study aimed at developing a sensitive fluorometric assay for detecting influenza viral RNA through tandem gene amplification methods including reverse transcription PCR (RT-PCR), followed by strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). Influenza viral RNA was initially amplified by RT-PCR with a tailed reverse primer containing an additional sequence for SDA. The RT-PCR amplicon was then subjected to SDA, yielding multiple copies of single-stranded DNA (ssDNA) that can be used as a primer for subsequent RCA. Thereafter, a long ssDNA segment harboring tandem repeated G-quadruplexes that were generated through RCA was intercalated by Thioflavin T, yielding a strong fluorescence signal indicating the presence of the target viral RNA. Fluorometric analysis detected influenza viral RNA ranging from 50 pg to 500 pg with a limit of detection of 6.2 pg with a signal-to-background ratio of 10 and identified each influenza virus strain (H1N1, H3N2, and influenza B). Thus, the present method for the label-free fluorometric detection of viral RNA via tandem gene amplifications combining RT-PCR-coupled SDA and G-quadruplex-generating RCA would facilitate the efficient diagnosis of influenza infection. Label-free fluorometric detection of influenza viral RNA by strand displacement coupled with rolling circle amplification.
ISSN:0003-2654
1364-5528
DOI:10.1039/d0an01326a