Karunaithas S.Chaibun T.Rijiravanich P.Puenpa J.Poovorawan Y.Yin L.S.Promptmas C.Athamanolap P.Lertanantawong B.Mahidol University2026-02-062026-02-062026-01-01Sensors and Actuators B Chemical Vol.446 (2026)09254005https://repository.li.mahidol.ac.th/handle/123456789/114732Rapid and accurate diagnosis of infectious diseases is of paramount importance for effective prevention and treatment. The recent COVID-19 pandemic accelerated the development of on-site detection tools for rapid screening and diagnosis. Recombinase polymerase amplification (RPA) is a well-established isothermal amplification method that offers a potential alternative to polymerase chain reaction for point-of-care applications. However, RPA's specificity remains limited due to non-specific amplification. In this study, we developed a betaine-assisted multiplex RPA system coupled with lateral flow assay for the simultaneous detection and typing of human respiratory virus variants, addressing the issue of non-specific amplification. Our results demonstrated that the addition of 8 µL of betaine per reaction effectively eliminated non-specific amplification in the multiplex RPA system. This method was applied to detect SARS-CoV-2 variants, and its analytical and clinical performance was evaluated. The findings revealed that betaine-assisted multiplex RPA specifically detects SARS-CoV-2 variants with a limit of detection of 1 fM, visible to the naked eye. Furthermore, 120 clinical samples, which included negative, alpha variant, and delta variant cases, were tested and showed 100 % concordance with the standard RT-qPCR method. This proof of concept can be adapted for the detection of various pathogens, particularly for screening emerging variants during outbreaks.Materials ScienceChemistryPhysics and AstronomyEngineeringArticleSCOPUS10.1016/j.snb.2025.1387422-s2.0-105015507213