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Comparison of paper-based nucleic acid extraction materials for point-of-care testing applications

ABSTRACT: Cheap, rapid, simple and equipment-free nucleic acid extraction (NAE) is highly preferred for implementing nucleic acid detection at point-of-care (POC). Paper-based NAE materials have been extensively utilized due to their low cost, abundance, portability, biocompatibility and ease of chemical modification. However, it is challenging for users to choose the proper one from existing paper-based NAE materials for specific POC applications, which is determined by their physical and chemical properties. Additionally, building the relationship between the physical and chemical properties and the NAE efficiency of paper-based materials is instructive for development of new paper-based NAE materials. In this study, we first systematically compared the physical and chemical properties of six widely used paper-based NAE materials (namely Whatman filter paper #1, FTA card, FTA elute card, Fusion 5, silica membrane and polyethersulfone (PES) membrane), and then evaluated their NAE efficiency. The obtained results indicated that pore uniformity, wet strength, porosity and functional groups are key parameters to affect the efficiency of NAE. The NAE performance of FTA card is the best with high concentration and purity. Finally, we envision that more cost-effective paper-based NAE materials will be developed for POCT application in the future.

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Supplementary Information

The online version contains supplementary material available at 10.1007/s10570-022-04444-6.

SUBMITTER: Tang R

PROVIDER: S-EPMC8799969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Nearly 7% of the world's population live with a hemoglobin variant. Hemoglobins S, C, and E are the most common and significant hemoglobin variants worldwide. Sickle cell disease, caused by hemoglobin S, is highly prevalent in sub-Saharan Africa and in tribal populations of Central India. Hemoglobin C is common in West Africa, and hemoglobin E is common in Southeast Asia. Screening for significant hemoglobin disorders is not currently feasible in many low-income countries with the high disease burden. Lack of early diagnosis leads to preventable high morbidity and mortality in children born with hemoglobin variants in low-resource settings. Here, we describe HemeChip, the first miniaturized, paper-based, microchip electrophoresis platform for identifying the most common hemoglobin variants easily and affordably at the point-of-care in low-resource settings. HemeChip test works with a drop of blood. HemeChip system guides the user step-by-step through the test procedure with animated on-screen instructions. Hemoglobin identification and quantification is automatically performed, and hemoglobin types and percentages are displayed in an easily understandable, objective way. We show the feasibility and high accuracy of HemeChip via testing 768 subjects by clinical sites in the United States, Central India, sub-Saharan Africa, and Southeast Asia. Validation studies include hemoglobin E testing in Bangkok, Thailand, and hemoglobin S testing in Chhattisgarh, India, and in Kano, Nigeria, where the sickle cell disease burden is the highest in the world. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. Study design, methods, and results are presented according to the Standards for Reporting Diagnostic Accuracy (STARD). HemeChip correctly identified all subjects with hemoglobin S, C, and E variants with 100% sensitivity, and displayed an overall diagnostic accuracy of 98.4% in comparison to reference standard methods. HemeChip is a versatile, mass-producible microchip electrophoresis platform that addresses a major unmet need of decentralized hemoglobin analysis in resource-limited settings.

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Comparison of paper-based nucleic acid extraction materials for point-of-care testing applications

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Supplementary Information

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