Evaluation of amplicons by AF4 as assistant for deep comprehension of loop-mediated isothermal amplification combined with lateral flow assay.

Abstract

Loop-mediated isothermal amplification (LAMP) is a rapid and efficient method for DNA amplification, producing concatemers of varying lengths (amplicons). This study explores the characterization of LAMP amplicons using asymmetric flow field-flow fractionation (AF4) and their realization in LAMP - lateral flow assay (LFA) for point-of-care diagnostics. We examined LAMP products from the invA gene of Salmonella enterica using two specific primer sets and three methods: fluorescent staining with SYBR Green, electrophoretic detection, and AF4. LAMP products labeled with biotin and fluorescein (introduced through primers, 12 combinations) were detected by LFA due to binding the labels. The efficiency of amplicon detection by test strips varied depending on the primer combination: the color intensities for the best and worst combinations differed by more than 7 times. AF4 enabled precise detection of components from primers to amplicons up to 500 bp and revealed differences influenced by labeling and primer combinations. An important advantage of AF4, which was demonstrated for the first time, was the characterization of native complexes of amplicons with receptors. The analysis of amplicons in complexes with streptavidin, anti-fluorescein antibodies, or both receptors together using AF4 revealed significant differences in the fractograms. The shape and maxima of the absorption peaks at 260 nm depended on retention time in the range from 20 to 35 min, indicating the formation of complexes of different compositions. AF4 and LFA showed that smaller amplicons (200-300 bp) primarily contribute to the binding of detected labels, while longer amplicons (1000-1500 bp) do not enhance the signal due to structural complexities. Our findings demonstrate the potential of AF4 as a tool for deep comprehension of LAMP processes and the composition of the formed amplicon-receptor complexes, offering valuable insights for enhancing LAMP-LFA systems. This work provides a foundation for the advanced application of AF4 for nucleic acid amplification technologies.