Near-digital amplification in paper improves sensitivity and speed in biplexed reactions
Abstract 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 wel...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2022-08-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-18937-8 |
| _version_ | 1811285877612085248 |
|---|---|
| author | Kamal G. Shah Sujatha Kumar Paul Yager |
| author_facet | Kamal G. Shah Sujatha Kumar Paul Yager |
| author_sort | Kamal G. Shah |
| collection | DOAJ |
| description | Abstract 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 103 copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 105 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. |
| first_indexed | 2024-04-13T02:51:22Z |
| format | Article |
| id | doaj.art-86e407578da248b6b215f73fe52c0d93 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-13T02:51:22Z |
| publishDate | 2022-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-86e407578da248b6b215f73fe52c0d932022-12-22T03:05:50ZengNature PortfolioScientific Reports2045-23222022-08-0112111010.1038/s41598-022-18937-8Near-digital amplification in paper improves sensitivity and speed in biplexed reactionsKamal G. Shah0Sujatha Kumar1Paul Yager2Department of Bioengineering, University of WashingtonDepartment of Bioengineering, University of WashingtonDepartment of Bioengineering, University of WashingtonAbstract 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 103 copies of methicillin-resistant Staphylococcus aureus genomic DNA to between 100 and 316 copies in a biplexed reaction containing 105 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.https://doi.org/10.1038/s41598-022-18937-8 |
| spellingShingle | Kamal G. Shah Sujatha Kumar Paul Yager Near-digital amplification in paper improves sensitivity and speed in biplexed reactions Scientific Reports |
| 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 |
| url | https://doi.org/10.1038/s41598-022-18937-8 |
| work_keys_str_mv | AT kamalgshah neardigitalamplificationinpaperimprovessensitivityandspeedinbiplexedreactions AT sujathakumar neardigitalamplificationinpaperimprovessensitivityandspeedinbiplexedreactions AT paulyager neardigitalamplificationinpaperimprovessensitivityandspeedinbiplexedreactions |