Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout
| Autor: | Kun Yin, Vikram Pandian, Zeinab H. Helal, Xiong Ding, Ziyue Li, Joan A. Smyth, Changchun Liu |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
DNA Bacterial Pathogen detection Loop-mediated isothermal amplification Oligonucleotides lcsh:Medicine 01 natural sciences Sensitivity and Specificity Article Fluorescence 03 medical and health sciences chemistry.chemical_compound Ribonucleases Ticks Animals Humans Borrelia burgdorferi lcsh:Science Analytical biochemistry Sensors and probes Lyme Disease Multidisciplinary biology Oligonucleotide Assay systems lcsh:R 010401 analytical chemistry Ribonucleotides Dna amplification biology.organism_classification 0104 chemical sciences Rec A Recombinases 030104 developmental biology chemistry Molecular Diagnostic Techniques Point-of-Care Testing Biophysics Nucleic acid lcsh:Q Smartphone Biomedical engineering Nucleic Acid Amplification Techniques DNA |
| Zdroj: | Scientific Reports Scientific Reports, Vol 10, Iss 1, Pp 1-9 (2020) |
| ISSN: | 2045-2322 |
| Popis: | Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings. |
| Databáze: | OpenAIRE |
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