Point-of-Care Diagnosis of Malaria Using a Simple, Purification-Free DNA Extraction Method Coupled with Loop-Mediated Isothermal Amplification-Lateral Flow

Abstract

We propose a protocol suitable for point-of-care diagnosis of malaria utilizing a simple and purification-free DNA extraction method with the combination of loop-mediated isothermal amplification assay and lateral flow (LAMP-LF). The multiplex LAMP-LF platform developed here can simultaneously detect Plasmodium knowlesi, P. vivax, P. falciparum, and Plasmodium genus (for P. malariae and P. ovale). Through the capillary effect, the results can be observed by the red band signal on the test and control lines within 5 min. The developed multiplex LAMP-LF was tested with 86 clinical blood samples on-site at Hospital Kapit, Sarawak, Malaysia. By using microscopy as the reference method, the multiplex LAMP-LF showed 100% sensitivity (95% confidence interval (CI): 91.4 to 100.00%) and 97.8% specificity (95% CI: 88.2% to 99.9%). The high sensitivity and specificity of multiplex LAMP-LF make it ideal for use as a point-of-care diagnostic tool. The simple and purification-free DNA extraction protocol can be employed as an alternative DNA extraction method for malaria diagnosis in resource-limited settings. By combining the simple DNA extraction protocol and multiplex LAMP-LF approach, we aim to develop a simple-to-handle and easy-to-read molecular diagnostic tool for malaria in both laboratory and on-site settings.

Advancements and applications of loop-mediated isothermal amplification technology: a comprehensive overview

“…The lateral flow assay (LFA) is a simple, cheap, user-friendly method based on an immunochromatographic technique without any special equipment for analysis, making it one of the most popular techniques for POCT. Lai et al (2023) developed a LAMP-Lateral Flow (LAMP-LF) test strip, which includes various components such as a conjugate pad with streptavidin-bound gold nanoparticles, test lines containing anti-fluorescein isothiocyanate, anti-digoxigenin, anti-cyanine5, anti-dinitrophenol, and a control line with biotinylated-BSA. The detection of four targets, including Plasmodium spp.-LAMP product, P. falciparum -LAMP product, P. vivax -LAMP product, and P. knowlesi -LAMP product, is achieved through antigen–antibody reactions.…”

Point-of-care testing of infectious diseases: recent advances

“…To this end, Lai et al developed a multiplex LAMP reaction system, and combined with a homemade multiplex lateral flow strip for the simultaneous detection of P. falciparum , P. vivax , Plasmodium malariae ( P. malariae ), Plasmodium ovale ( P. ovale ) and Plasmodium knowlesi ( P. knowlesi ). 115 The DNA extraction protocol without purification was applied to shorten the time and better suit resource-limited areas. With microscopy as the reference method, this established method tested 86 clinical samples with 100% sensitivity and 97.8% specificity.…”