A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

English version

Autoři: Florian Katzmeier ^aff001; Lukas Aufinger ^aff001; Aurore Dupin ^aff001; Jorge Quintero ^aff002; Matthias Lenz ^aff001; Ludwig Bauer ^aff001; Sven Klumpe ^aff001; Dawafuti Sherpa ^aff002; Benedikt Dürr ^aff002; Maximilian Honemann ^aff001; Igor Styazhkin ^aff001; Friedrich C. Simmel ^aff001; Michael Heymann ^aff003
Působiště autorů: Physics Department and ZNN, Technical University of Munich, Garching, Germany ^aff001; Department of Biology, Ludwig-Maximilians-Universität Munich, Martinsried, Germany ^aff002; Intelligent Biointegrative Systems Group, Institute for Biomaterials and Biomolecular Systems, University Stuttgart, Germany ^aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0220091

Souhrn

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

Klíčová slova:

Artificial light – Colorimetric assays – Filter paper – Fluorescence – Nucleic acids – Ribonucleases – Signal filtering – Signal processing

Zdroje

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A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

Souhrn

Klíčová slova:

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