Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse-transcription loop-mediated isothermal amplification

Abstract

AbstractAmid the enduring COVID-19 pandemic, there is an urgent need for expanded access to rapid and sensitive SARS-CoV-2 testing worldwide. Here we present a simple clinical workflow that uses a sensitive and highly specific colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to detect SARS-CoV-2 and takes forty minutes from sample collection to result. This test requires no specialized equipment and costs a few dollars per sample. Nasopharyngeal samples collected in saline were added either directly (unprocessed) to RT-LAMP reactions or first inactivated by a combined chemical and heat treatment step to inhibit RNases and lyse virions and human cells. The specimens were then amplified with two SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. While direct addition of unprocessed specimens to RT-LAMP reactions could reliably detect samples with abundant SARS-CoV-2, the assay sensitivity markedly increased after the addition of an inactivation step. In 62 clinical samples with a wide range of SARS-CoV-2 nucleic acid concentrations, the assay had 87.5% sensitivity and 100% specificity with a limit of detection at least 25 copies/μL, making it an ideal test to rule in infection. To increase sensitivity, samples that tested negative for SARS-CoV-2 by direct sample addition could be reflexed to a purification step, to increase the effective per-reaction sample input volume. In 40 purified samples, the assay yielded a 90% sensitivity and 100% specificity, with a limit of detection comparable to commercially available real-time PCR-based diagnostics that have received Emergency Use Authorization (EUA) from the FDA. This test for SARS-CoV-2 can be performed in a range of settings for a fraction of the price of other available tests, with limited equipment, and without relying on over-burdened supply chains to increase overall testing capacity.