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bioRxiv · 10.1101/2024.12.16.626200

DIYNAFLUOR: An Affordable DIY Plug-and-Play Nucleic Acid Fluorometer for eDNA Quantification in Resource Limited Settings

Anderson, W.·Antaw, F.·Kenny, S.·Rupani, H.·Khamis, R.·Constantin, N.·Kumar, V.·Gemmell, A.·Bell, C.·Trau, M.·Korbie, D.
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Abstract

Nucleic Acid (NA) fluorometry is widely employed for quantifying environmental DNA (eDNA) samples and their downstream DNA sequencing libraries, owing to its sensitivity, accuracy, and speed. However, the high cost of NA fluorometers presents a barrier to eDNA sequencing in resource limited settings (RLSs). For instance, at [~]$1.5-3.3k USD, current NA fluorometers present a greater capital cost than ONTs $1k USD portable MinION third-generation Nanopore sequencing platform. The collapse of international scientific device and consumable supply chains during the COVID-19 pandemic also highlighted the need for distributed manufacturing of molecular research tools to mitigate the impact of increased pricing to RLSs. To address these challenges, we have developed the "DIYNAFLUOR" (DIY Nucleic Acid FLUORometer), a portable, open-source, <$40 USD NA fluorometer, designed using readily available off-the-shelf components, simple 3D-printed parts, and plug-and-play, solder-free assembly. The DIYNAFLUOR was primarily designed to be compatible with the popular DNA-centric Qubit High Sensitivity (HS) and Broad Range (BR) assay kits. Notably, the DIYNAFLUOR demonstrated an in-assay Limit of Detection with the Qubit HS kit of 0.0028 ng/L, and an average absolute bias of 0.018 ng/L across a 0-10 ng/L working range using a 2-point linear calibration methodology. Device verification was performed by comparative measurements with a Qubit 4 fluorometer in a busy biotechnology laboratory, and build instructions were validated through assembly and qualification of three DIYNAFLUOR devices by researchers outside the primary design team. We also describe a custom "extreme"low-cost assay, <13{cents} USD per-measurement, that uses SYBR Safe dye to quantify DNA across a working range of 0-0.5 ng/L. This assay reports a lower sensitivity and accuracy than commercial kits but may be of use to RLSs in times of extreme resource constraints or as a teaching tool for STEM educators. To demonstrate its practical application for field-based eDNA analysis in RLSs, the DIYNAFLUOR was used to perform all quality control measurements throughout the preparation of a 16S and 18S metabarcoding library generated from eDNA extracted from Australian lake water, leading to the successful identification of Australian fauna via Nanopore sequencing. Finally, configurations of the DIYNAFLUOR for RNA and Protein quantification are briefly described. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/626200v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@28244dorg.highwire.dtl.DTLVardef@16749acorg.highwire.dtl.DTLVardef@15c31aorg.highwire.dtl.DTLVardef@bc03e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Anderson, W., Antaw, F., Kenny, S., Rupani, H., Khamis, R., Constantin, N., Kumar, V., Gemmell, A., Bell, C., Trau, M., Korbie, D.. 2024-12-16. DIYNAFLUOR: An Affordable DIY Plug-and-Play Nucleic Acid Fluorometer for eDNA Quantification in Resource Limited Settings. https://doi.org/10.1101/2024.12.16.626200

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