RESUMO
Amid 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/L, 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.
RESUMO
As the current SARS-CoV-2 pandemic spreads, the need for more diagnostic capabilities is great. In order to address this need, we have developed a highly sensitive RT-LAMP assay compatible with current reagents, that utilizes a colorimetric readout in as little as 30 minutes. In addition to this, we have developed an inexpensive pipeline to further increase sensitivity without requiring highly specialized equipment. A rapid inactivation protocol capable of inactivating virions, as well as endogenous nucleases, was also developed to increase sensitivity and sample stability. This protocol, combined with our RT-LAMP assay, has a sensitivity of at least 50 viral RNA copies per microliter in a sample. To further increase the sensitivity, a purification protocol compatible with this inactivation method was developed. The inactivation and purification protocol, combined with our RT-LAMP assay, brings the sensitivity to at least 1 viral RNA copy per microliter in a sample. We hope that this inactivation and purification pipeline, which costs approximately $0.07 per sample and which uses readily available reagents, will increase the availability of SARS-CoV-2 testing, as well as expand the settings in which this testing can be performed.
