A dual paper-based nucleic acid extraction method from blood in under ten minutes for point-of-care diagnostics
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Author(s)
Malpartida-Cardenas, Kenny
Baum, Jake
Cunnington, Aubrey
Georgiou, Pantelis
Rodriguez-Manzano, Jesus
Type
Journal Article
Abstract
Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.
Date Issued
2023-07-07
Date Acceptance
2023-05-28
Citation
The Analyst, 2023, 148 (13), pp.3036-3044
ISSN
0003-2654
Publisher
Royal Society of Chemistry
Start Page
3036
End Page
3044
Journal / Book Title
The Analyst
Volume
148
Issue
13
Copyright Statement
© The Royal Society of Chemistry 2023. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/37265396
Publication Status
Published
Coverage Spatial
England
Date Publish Online
2023-06-02