Multiplex Target-Redundant RT-LAMP for Robust Detection of SARS-CoV-2 Using Fluorescent Universal Displacement Probes

Abstract

The increasing prevalence of variant lineages during the COVID-19 pandemic has the potential to disrupt molecular diagnostics due to mismatches between primers and variant templates. Point-of-care molecular diagnostics, which often lack the complete functionality of their high-throughput laboratory counterparts, are particularly susceptible to this type of disruption, which can result in false-negative results. To address this challenge, we have developed a robust Loop Mediated Isothermal Amplification assay with single tube multiplexed multitarget redundancy and an internal amplification control. A convenient and cost-effective target-specific fluorescence detection system allows amplifications to be grouped by signal using adaptable probes for pooled reporting of SARS-CoV-2 target amplifications or differentiation of the Internal Amplification Control. Over the course of the pandemic, primer coverage of viral lineages by the three redundant sub-assays has varied from assay to assay as they have diverged from the Wuhan-Hu-1 isolate sequence, but aggregate coverage has remained high for all variant sequences analyzed, with a minimum of 97.4% (Variant of Interest: Eta). In three instances (Delta, Gamma, Eta), a high-frequency mismatch with one of the three sub-assays was observed, but overall coverage remained high due to multitarget redundancy. When challenged with extracted human samples the multiplex assay showed 87% or better sensitivity (of 30 positive samples), with 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction (of 21 positive samples), and 100% specificity (of 60 negative samples). These results are further evidence that conventional laboratory methodologies can be leveraged at the point of care for robust performance and diagnostic stability over time. <b>IMPORTANCE</b> The COVID-19 pandemic has had tremendous impact, and the ability to perform molecular diagnostics in resource limited settings has emerged as a key resource for mitigating spread of the disease. One challenge in COVID-19 diagnosis, as well as other viruses, is ongoing mutation that can allow viruses to evade detection by diagnostic tests. We developed a test that detects multiple parts of the virus genome in a single test to reduce the chance of missing a virus due to mutation, and it is designed to be simpler and faster than typical laboratory tests while maintaining high sensitivity. This capability is enabled by a novel fluorescent probe technology that works with a simple constant temperature reaction condition.