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

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 requ...

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Autores principales: Katzmeier, Florian, Aufinger, Lukas, Dupin, Aurore, Quintero, Jorge, Lenz, Matthias, Bauer, Ludwig, Klumpe, Sven, Sherpa, Dawafuti, Dürr, Benedikt, Honemann, Maximilian, Styazhkin, Igor, Simmel, Friedrich C., Heymann, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919979/
https://www.ncbi.nlm.nih.gov/pubmed/31851676
http://dx.doi.org/10.1371/journal.pone.0220091
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author Katzmeier, Florian
Aufinger, Lukas
Dupin, Aurore
Quintero, Jorge
Lenz, Matthias
Bauer, Ludwig
Klumpe, Sven
Sherpa, Dawafuti
Dürr, Benedikt
Honemann, Maximilian
Styazhkin, Igor
Simmel, Friedrich C.
Heymann, Michael
author_facet Katzmeier, Florian
Aufinger, Lukas
Dupin, Aurore
Quintero, Jorge
Lenz, Matthias
Bauer, Ludwig
Klumpe, Sven
Sherpa, Dawafuti
Dürr, Benedikt
Honemann, Maximilian
Styazhkin, Igor
Simmel, Friedrich C.
Heymann, Michael
author_sort Katzmeier, Florian
collection PubMed
description 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.
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spelling pubmed-69199792020-01-07 A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a Katzmeier, Florian Aufinger, Lukas Dupin, Aurore Quintero, Jorge Lenz, Matthias Bauer, Ludwig Klumpe, Sven Sherpa, Dawafuti Dürr, Benedikt Honemann, Maximilian Styazhkin, Igor Simmel, Friedrich C. Heymann, Michael PLoS One Research Article 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. Public Library of Science 2019-12-18 /pmc/articles/PMC6919979/ /pubmed/31851676 http://dx.doi.org/10.1371/journal.pone.0220091 Text en © 2019 Katzmeier et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Katzmeier, Florian
Aufinger, Lukas
Dupin, Aurore
Quintero, Jorge
Lenz, Matthias
Bauer, Ludwig
Klumpe, Sven
Sherpa, Dawafuti
Dürr, Benedikt
Honemann, Maximilian
Styazhkin, Igor
Simmel, Friedrich C.
Heymann, Michael
A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title_full A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title_fullStr A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title_full_unstemmed A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title_short A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a
title_sort low-cost fluorescence reader for in vitro transcription and nucleic acid detection with cas13a
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919979/
https://www.ncbi.nlm.nih.gov/pubmed/31851676
http://dx.doi.org/10.1371/journal.pone.0220091
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