3’ end hydrolysis activity of the Bst polymerase enables rapid and sensitive identification of SARS-CoV-2 variants

Abstract Sensitive and accurate detection of genetic variants is important in many clinical applications, including the identification of specific pathogens or viral subtypes. However, existing methods are too time-consuming or complex, hampering their deployment in point-of-care settings. Here, we demonstrate that the intrinsic 3’ hydrolysis activity of the Bst polymerase deployed in loop-mediated isothermal amplification (LAMP) reactions can cleave off an end-conjugated quencher from a fully hybridised DNA oligonucleotide, allowing us to develop a fluorescent probe-based method that is highly sensitive to single nucleotide mismatches. We utilize our method, named SNiPER, to build diagnostic assays that detect different SARS-CoV-2 variants, including Alpha, Beta, Delta, and Omicron. Furthermore, we manufacture a simple device with 3D printing and inexpensive off-the-shelf electronics components to enable point-of-care testing and validate our assays on 32 patient samples, achieving perfect concordance with sequencing-based variant classification. Our technology can be quickly reconfigured to detect different pathogens and their variants, providing us with a useful tool to address any future infectious disease outbreak.