期刊
SENSORS AND ACTUATORS B-CHEMICAL
卷 251, 期 -, 页码 894-901出版社
ELSEVIER SCIENCE SA
DOI: 10.1016/j.snb.2017.05.101
关键词
Influenza viral RNA; Rolling circle amplification; G-quadruplex; Thioflavin T
资金
- Ministry of Health and Welfare, Republic of Korea [HI15C2917]
We developed a label-free fluorometric system based on double amplification of target nucleic acid by rolling circle amplification generating G-quadruplex (GQ-RCA) for sensitive detection of influenza virus RNA. To detect viral RNA with high sensitivity and selectivity, double amplification system was employed through reverse transcription PCR (RT-PCR) coupled in tandem with GQ-RCA. Single-stranded amplified viral DNA (ssDNA) was generated by RT-PCR of samples containing influenza virus RNA. The phosphate primed DNA strand in amplicon DNA was subsequently degraded by lambda exonuclease. Viral ssDNA formed a ternary RCA initiation complex by annealing to both a partial hairpin primer and a dumbbell padlock DNA template. A long stretch of ssDNA containing repeated copies of the G-quadruplex sequence was generated by RCA at room temperature. Sensitive detection of amplified target nucleic acid was then accomplished by monitoring fluorogenic interactions between Thioflavin T (ThT) and RCA-responsive G-quadruplexes. Differences in fluorescence intensity between target and non-target viral RNA were evident under UV illumination. Selective fluorescence staining of RCA-responsive G-quadruplexes enabled influenza virus RNA detection at concentrations as low as 4.9 aM with a linear detection range between 450 aM and 450 fM. The RT-PCR-coupled GQ-RCA system for influenza virus genome detection exhibited very high sensitivity and allowed convenient multiplexed virus detection within 2.5 h. Thus, a combination of RT-PCR-coupled GQ-RCA and ThT fluorescence staining can be a sensitive and accurate method for detecting RNA molecules of influenza viruses as well as those of other viruses. (C) 2017 Elsevier B.V. All rights reserved.
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