B-213 Colorimetric LAMP as an Effective Tool for Rapid Molecular Diagnostics

Abstract Background LAMP has become a standard molecular diagnostic technique, and its robustness and flexibility have enabled rapid, simple tests outside of traditional laboratory settings. The need for additional diagnostic methods became urgent during the SARS-CoV-2 pandemic, and LAMP was extensively evaluated and validated for numerous testing applications. We have extended the utility of LAMP to enable colorimetric detection, multiplex assays, variant identification, and robust direct tests for point of-care and workplace surveillance testing. Methods We utilized fluorescent and colorimetric RT-LAMP for assay development and our workplace screening program. A simple lysis buffer was applied to whole saliva samples and added directly to our colorimetric LAMP SARS-CoV-2 tests, with more than 100 000 samples tested. For variant calling we developed novel hybridization probes with single-base sensitivity to regions of sequence changes, and we characterized more than 5000 LAMP assays internally positioning mismatches in the LAMP primer regions to ensure robustness to and tolerance of mutations. Results The colorimetric LAMP assay displayed a sensitivity of 97% and specificity of 100%, and during its utilization in our in-house CLIA-certified workplace screening has identified >300 unique positive cases derived from all variants of concern. 3654 blinded samples were analyzed by 3 operators and colorimetric readout was found to have a strong concordance with real-time fluorescence measurement (kappa = 0.999). Conclusion RT-LAMP is remarkably tolerant of sequence variation, with no individual positional mutation significantly affecting assay performance. Simple visual readout can be utilized for a reliable diagnostic indicator when sophisticated instrumentation is not available or desired.