Single universal primer recombinase polymerase amplification-based lateral flow biosensor (SUP-RPA-LFB) for multiplex detection of genetically modified maize.

In this study, an isothermal paper biosensor, combining single universal primer recombinase polymerase amplification (SUP-RPA) and the lateral flow technique was developed for the multiplex detection of genetically modified maize (GMM). In pre-amplification stage, the event-specific primers contain a universal sequence at the 5' end, with a biotin-labeled deoxycytidine triphosphate (dCTP) deoxynucleotide providing additional amplification, which improves their amplification ability and ensures consistent multiplex amplification efficiency. In the signal recognition strategy, the SUP-RPA products are identified visually using the lateral flow biosensor (LFB) through dual hybridization. The accumulation of gold nanoparticles (AuNPs) produces a characteristic red band. Through this biosensor, a limit of detection of at least 50 copies was achieved, which is sensitive enough to detect MON810, MON863 and MON89034 simultaneously. The entire process of analysis was completed within 30 min and without any large-scale instrumentation. This biosensor, therefore, provides a novel rapid and portable multiple detection method for point-of-care applications, especially genetically modified organism (GMO) event-specific detection.