Department of Bioscience and Biotechnology, Konkuk University, Gwangjin-gu, Seoul 05029, Republic of Korea.
Analyst. 2021 Jan 7;145(24):8002-8007. doi: 10.1039/d0an01326a.
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.
由于流感每年在全球范围内发生，因此开发一种简便、准确的方法来检测流感病毒非常重要。本研究旨在通过包括逆转录 PCR（RT-PCR）在内的串联基因扩增方法开发一种灵敏的荧光检测流感病毒 RNA 的方法，随后进行链置换扩增（SDA）和滚环扩增（RCA）。流感病毒 RNA 最初通过 RT-PCR 进行扩增，RT-PCR 采用带有额外 SDA 序列的长尾反转引物。然后将 RT-PCR 扩增子进行 SDA，产生多个单链 DNA（ssDNA）拷贝，可作为后续 RCA 的引物。随后，通过 RCA 生成的长 ssDNA 片段中串联重复的 G-四链体通过噻唑橙嵌入，产生强荧光信号，表明存在目标病毒 RNA。荧光分析检测到 50pg 至 500pg 的流感病毒 RNA，检测限为 6.2pg，信号背景比为 10，并鉴定出每种流感病毒株（H1N1、H3N2 和乙型流感）。因此，本研究通过结合 RT-PCR 偶联 SDA 和 G-四链体生成 RCA 的串联基因扩增，实现了无标记荧光检测病毒 RNA 的方法，将有助于流感感染的有效诊断。
