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Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design
Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10667681/ https://www.ncbi.nlm.nih.gov/pubmed/38028620 http://dx.doi.org/10.3389/fgene.2023.1272964 |
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author | Edwards, Rebecca L. Takach, Johanna E. McAndrew, Michael J. Menteer, Jondavid Lestz, Rachel M. Whitman, Douglas Baxter-Lowe, Lee Ann |
author_facet | Edwards, Rebecca L. Takach, Johanna E. McAndrew, Michael J. Menteer, Jondavid Lestz, Rachel M. Whitman, Douglas Baxter-Lowe, Lee Ann |
author_sort | Edwards, Rebecca L. |
collection | PubMed |
description | Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%–0.13%, and those for analytical limit of detection were 0.00%–0.20%. Inter-laboratory reproducibility was excellent (r (2) = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings. |
format | Online Article Text |
id | pubmed-10667681 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-106676812023-11-10 Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design Edwards, Rebecca L. Takach, Johanna E. McAndrew, Michael J. Menteer, Jondavid Lestz, Rachel M. Whitman, Douglas Baxter-Lowe, Lee Ann Front Genet Genetics Digital PCR (dPCR) is a powerful tool for research and diagnostic applications that require absolute quantification of target molecules or detection of rare events, but the number of nucleic acid targets that can be distinguished within an assay has limited its usefulness. For most dPCR systems, one target is detected per optical channel and the total number of targets is limited by the number of optical channels on the platform. Higher-order multiplexing has the potential to dramatically increase the usefulness of dPCR, especially in scenarios with limited sample. Other potential benefits of multiplexing include lower cost, additional information generated by more probes, and higher throughput. To address this unmet need, we developed a novel melt-based hairpin probe design to provide a robust option for multiplexing digital PCR. A prototype multiplex digital PCR (mdPCR) assay using three melt-based hairpin probes per optical channel in a 16-well microfluidic digital PCR platform accurately distinguished and quantified 12 nucleic acid targets per well. For samples with 10,000 human genome equivalents, the probe-specific ranges for limit of blank were 0.00%–0.13%, and those for analytical limit of detection were 0.00%–0.20%. Inter-laboratory reproducibility was excellent (r (2) = 0.997). Importantly, this novel melt-based hairpin probe design has potential to achieve multiplexing beyond the 12 targets/well of this prototype assay. This easy-to-use mdPCR technology with excellent performance characteristics has the potential to revolutionize the use of digital PCR in research and diagnostic settings. Frontiers Media S.A. 2023-11-10 /pmc/articles/PMC10667681/ /pubmed/38028620 http://dx.doi.org/10.3389/fgene.2023.1272964 Text en Copyright © 2023 Edwards, Takach, McAndrew, Menteer, Lestz, Whitman and Baxter-Lowe. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genetics Edwards, Rebecca L. Takach, Johanna E. McAndrew, Michael J. Menteer, Jondavid Lestz, Rachel M. Whitman, Douglas Baxter-Lowe, Lee Ann Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title | Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title_full | Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title_fullStr | Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title_full_unstemmed | Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title_short | Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design |
title_sort | next generation multiplexing for digital pcr using a novel melt-based hairpin probe design |
topic | Genetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10667681/ https://www.ncbi.nlm.nih.gov/pubmed/38028620 http://dx.doi.org/10.3389/fgene.2023.1272964 |
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