Betaine-assisted multiplex recombinase polymerase amplification coupled lateral flow assay for simultaneous detection and typing of variants in human respiratory virus

Rapid 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.