Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings
| Autor: | Mary E. Natoli, Rebecca Richards-Kortum, Yajur Maker, Kathryn Kundrod, Alex Kortum, Imran Vohra, Jackson Coole, Yubo Tang |
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| Rok vydání: | 2021 |
| Předmět: |
Materials science
General Chemical Engineering Loop-mediated isothermal amplification Recombinase Polymerase Amplification 010402 general chemistry Real-Time Polymerase Chain Reaction 01 natural sciences Isothermal process General Biochemistry Genetics and Molecular Biology Fluorometer Nucleic Acids Humans Chromatography General Immunology and Microbiology SARS-CoV-2 General Neuroscience 010401 analytical chemistry COVID-19 Nucleic acid amplification technique Reverse Transcription Amplicon 0104 chemical sciences Real-time polymerase chain reaction Molecular Diagnostic Techniques Printing Three-Dimensional Nucleic acid Health Resources RNA Viral Nucleic Acid Amplification Techniques |
| Zdroj: | Journal of visualized experiments : JoVE. (168) |
| ISSN: | 1940-087X |
| Popis: | Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA. |
| Databáze: | OpenAIRE |
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