Comparison and Optimization of Simple DNA Extraction Methods for LAMP-Based Point-of-Care Applications Employing...

Loop-mediated isothermal amplification (LAMP) has gained particular attention for point-of-care (POC) applications due to its advantages over traditional nucleic acid testing approaches. However, a prevailing limitation of LAMP in POC applications is nucleic acid extraction from the sample prior to analysis. This is particularly true for complex samples such as submillimeter skin biopsies where lysis and digestion involve intricate and lengthy procedures. The objective of this study was to compare alternative methodologies against the spin-column laboratory standard and evaluate them based on the World Health Organization ASSURED criteria for POC testing. Four methods-magnetic bead extraction, alkaline extraction, proteinase K-heat inactivation extraction, and boiling method extraction-were optimized utilizing porcine skin submillimeter punch biopsies and subsequently validated on human skin. Results show that both alkaline extraction and proteinase K-heat inactivation produce DNA yields equivalent to or higher than the spin-column method in porcine and human skin. When evaluated against the ASSURED criteria, both methods demonstrated low complexity while being highly scalable and readily accessible. Overall, this comparative study established a robust framework for selecting DNA extraction methods for submillimeter skin biopsies in POC applications. It also underscored the performance of the alkaline extraction method based on the ASSURED criteria, providing equivalent DNA yields to laboratory standards with reduced complexity and potential for cost-effective scalability.