RESUMEN
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 multi-target 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 multi-target redundancy. When challenged with extracted human samples the multiplexed assay showed 100% sensitivity for samples containing greater than 30 copies of viral RNA per reaction, and 100% specificity. 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.
RESUMEN
RNA amplification tests allow sensitive detection of SARS-CoV-2 infection, but their complexity and cost are prohibitive for expanding COVID-19 testing. We developed "Harmony COVID-19", a point-of-care test using inexpensive consumables, ready-to-use reagents, and a simple device that processes up to 4 samples simultaneously. Our lyophilized reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) can detect as little as 15 SARS-CoV-2 RNA copies per reaction, and it can report as early as 17 min for samples with high viral load (2 x 105 RNA copies per reaction). Analysis of RNA extracted from clinical nasal specimens (n = 101) showed 95% concordance with RT-PCR, including 100% specificity in specimens positive for other viruses and bacteria. Analysis of contrived samples in the nasal matrix showed detection of 92% or 100% in samples with [≥]20 or [≥]100 particles per reaction, respectively. Usability testing showed 95% accuracy by healthcare workers operating the test for the first time. ONE SENTENCE SUMMARYHarmony COVID-19: point-of-care SARS-CoV-2 RNA detection