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Near-digital amplification in paper improves sensitivity and speed in biplexed reactions

The simplest point-of-care assays are usually paper and plastic devices that detect proteins or nucleic acids at low cost and minimal user steps, albeit with poor limits of detection. Digital assays improve limits of detection and analyte quantification by splitting a sample across many wells (or dr...

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Autores principales: Shah, Kamal G., Kumar, Sujatha, Yager, Paul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9418329/
https://www.ncbi.nlm.nih.gov/pubmed/36028745
http://dx.doi.org/10.1038/s41598-022-18937-8
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author Shah, Kamal G.
Kumar, Sujatha
Yager, Paul
author_facet Shah, Kamal G.
Kumar, Sujatha
Yager, Paul
author_sort Shah, Kamal G.
collection PubMed
description The simplest point-of-care assays are usually paper and plastic devices that detect proteins or nucleic acids at low cost and minimal user steps, albeit with poor limits of detection. Digital assays improve limits of detection and analyte quantification by splitting a sample across many wells (or droplets), preventing diffusion, and performing analyte amplification and detection in multiple small wells. However, truly digital nucleic acid amplification tests (NAATs) require costly consumable cartridges that are precisely manufactured, aligned, and operated to enable low detection limits. In this study, we demonstrate how to implement near-digital NAATs in low-cost porous media while approaching the low limits of detection of digital assays. The near-digital NAAT was enabled by a paper membrane containing lyophilized amplification reagents that automatically, passively meters and distributes a sample over a wide area. Performing a NAAT in the paper membrane while allowing diffusion captures many of the benefits of digital NAATs if the pad is imaged at a high spatial resolution during amplification. We show that the near-digital NAAT is compatible with a low-cost paper and plastic disposable cartridge coupled to a 2-layer rigid printed circuit board heater (the MD NAAT platform). We also demonstrate compatibility with biplexing and imaging with mobile phones with different camera sensors. We show that the near-digital NAAT increased signal-to-noise ratios by ~ 10×, improved limits of detection from above 10(3) copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 10(5) copies of co-amplifying internal amplification control DNA, and reduced time-to-result from 45 min of amplification to 15–20 min for the positive samples.
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spelling pubmed-94183292022-08-28 Near-digital amplification in paper improves sensitivity and speed in biplexed reactions Shah, Kamal G. Kumar, Sujatha Yager, Paul Sci Rep Article The simplest point-of-care assays are usually paper and plastic devices that detect proteins or nucleic acids at low cost and minimal user steps, albeit with poor limits of detection. Digital assays improve limits of detection and analyte quantification by splitting a sample across many wells (or droplets), preventing diffusion, and performing analyte amplification and detection in multiple small wells. However, truly digital nucleic acid amplification tests (NAATs) require costly consumable cartridges that are precisely manufactured, aligned, and operated to enable low detection limits. In this study, we demonstrate how to implement near-digital NAATs in low-cost porous media while approaching the low limits of detection of digital assays. The near-digital NAAT was enabled by a paper membrane containing lyophilized amplification reagents that automatically, passively meters and distributes a sample over a wide area. Performing a NAAT in the paper membrane while allowing diffusion captures many of the benefits of digital NAATs if the pad is imaged at a high spatial resolution during amplification. We show that the near-digital NAAT is compatible with a low-cost paper and plastic disposable cartridge coupled to a 2-layer rigid printed circuit board heater (the MD NAAT platform). We also demonstrate compatibility with biplexing and imaging with mobile phones with different camera sensors. We show that the near-digital NAAT increased signal-to-noise ratios by ~ 10×, improved limits of detection from above 10(3) copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 10(5) copies of co-amplifying internal amplification control DNA, and reduced time-to-result from 45 min of amplification to 15–20 min for the positive samples. Nature Publishing Group UK 2022-08-26 /pmc/articles/PMC9418329/ /pubmed/36028745 http://dx.doi.org/10.1038/s41598-022-18937-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Shah, Kamal G.
Kumar, Sujatha
Yager, Paul
Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title_full Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title_fullStr Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title_full_unstemmed Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title_short Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
title_sort near-digital amplification in paper improves sensitivity and speed in biplexed reactions
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9418329/
https://www.ncbi.nlm.nih.gov/pubmed/36028745
http://dx.doi.org/10.1038/s41598-022-18937-8
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