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

Abstract

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 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) : medRxiv preprint SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted.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.

SARS-CoV-2 detection using isothermal amplification and a rapid, inexpensive protocol for sample inactivation and purification

“…The current sample collection methods used for SARS-CoV-2 testing require swabs to be placed in 2 mL to 3 mL of commercial collection media, such as Quest Diagnostics Viral Collection Media (VCM) ( 7 ). This method presents a serious challenge for RT-LAMP-based detection, as very little (no more than 1 µL) can be used in a 25-µL reaction, due to the presence of dyes and buffers in the VCM that would prevent visualization of a pH shift in a positive reaction ( 8 ). Other collection media, such as 0.9% saline, have less of an inhibitory effect.…”

“…Glass milk purification increases this sensitivity at least 10-fold. Furthermore, the specificity was found to be 100%, with no false positives reported (8).…”

“…Furthermore, if a genomic mutation were to prevent one assay from returning a positive result, a second assay targeting a different region of the genome could still detect the viral RNA. It is also important that tests used for patient samples include an internal control, such as beta-actin used in the clinical validation of this assay (8), to ensure that RNA integrity in the sample is maintained and a negative result is not due to degradation.…”

“…When used with patient samples, Orf1a-HMSe performed very well, with inactivation greatly improving the limit of detection (8). Inactivation and use of the Orf1a-HMSe primers provided for a sensitivity of ∼85% of the currently used qRT-PCR assay, with robust sensitivity (>95%) for samples with Ct values under 30, corresponding to samples with ∼40 virions per microliter.…”

“…The overall cost of the inactivation and purification is approximately $0.07 per sample. Early clinical validation of these protocols with patient samples consisting of swabs in saline has shown that inactivation alone increases the sensitivity to >95% of samples with qRT-PCR Ct values of <30, and 85% sensitivity overall (8). Glass milk purification increases this sensitivity at least 10-fold.…”