Efficient multiplexing and variant discrimination in reverse-transcription loop-mediated isothermal amplification with sequence-specific hybridization probes.

Loop-mediated isothermal amplification (LAMP) has proven a robust and reliable nucleic acid amplification method that is well suited for simplified and rapid molecular diagnostics. Various approaches have emerged for sequence-specific detection of LAMP products, but with limitations to their widespread utility or applicability for single-nucleotide polymorphism detection and multiplexing. Here we demonstrate the use of simple hybridization probes (as used for qPCR) that enable simple multiplexing and SARS-CoV-2 variant typing in reverse-transcription LAMP. This approach requires no modification to the LAMP primers and is amenable to the detection of single-nucleotide polymorphisms and small sequence changes, which is usually difficult in LAMP. By extending LAMP's ability to be utilized for multitarget and single-base change detection, we hope to increase its potential to enable more and better molecular diagnostic testing.