Real-time optical analysis of a colorimetric LAMP assay for SARS-CoV-2 in saliva with a handheld instrument improves accuracy compared to endpoint assessment

Abstract

Controlling the course of the COVID-19 pandemic will require widespread deployment of consistent and accurate diagnostic testing of the novel coronavirus SARS-CoV-2. Ideally, tests should detect a minimum viral load, be minimally invasive, and provide a rapid and simple readout. Current FDA-approved RT-qPCR-based standard diagnostic approaches require invasive nasopharyngeal swabs and involve laboratory-based analyses that can delay results. Recently, a loop mediated isothermal nucleic acid amplification (LAMP) test that utilizes colorimetric readout received FDA approval. This approach utilizes a pH indicator dye to detect drop in pH from nucleotide hydrolysis during nucleic acid amplification. This method has only been approved for use with RNA extracted from clinical specimens collected via nasopharyngeal swabs. In this study, we developed a quantitative LAMP-based strategy to detect SARS-CoV-2 RNA in saliva. Our detection system distinguished positive from negative sample types using a handheld instrument that monitors optical changes throughout the LAMP reaction. We used this system in a streamlined LAMP testing protocol that could be completed in less than two hours to directly detect inactivated SARS-CoV-2 in minimally processed saliva that bypassed RNA extraction, with a limit of detection (LOD) of 50 genomes/reaction. The quantitative method correctly detected virus in 100% of contrived clinical samples spiked with inactivated SARS- CoV-2 at either 1X (50 genomes/reaction) or 2X (100 genomes/reaction) of the LOD. Importantly the quantitative method was based on dynamic optical changes during the reaction so was able to correctly classify samples that were misclassified by endpoint observation of color.

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy

“…However, modifications of RT-LAMP-based SARS-CoV-2 assays to reduce saliva sample variability, improve result ambiguity, and increase throughput have recently been reported elsewhere and may improve the implementation of RT-LAMP-based assays for POC use. [46][47][48][49][50] In addition, we relied on a manual RT-LAMP format during POC testing. Reading assays "by eye" inevitably results in a somewhat-subjective determination of positives.…”

Detection of SARS-CoV-2 Virus Amplification Using a Crumpled Graphene Field-Effect Transistor Biosensor

“…Since the current pandemic brought the point-of-care (POC) diagnostic tests into the spotlight, many POC devices for the diagnosis of COVID-19 have been reported in the literature or have been awarded an Emergency Use Authorization (EUA) over the past year. Although most of them rely on visual readout techniques, ,− several electrical readout options are also available. Electrical readout options reduce cost by avoiding the necessity of fluorophores, lasers, LEDs, cameras, etc.…”

A robust, low-cost instrument for real-time colorimetric isothermal nucleic acid amplification

“…However, sample plates are heated via convection, which increases assay time and raises the risk of nonspecific amplification at intermediate temperatures as the assay warms up. Researchers have demonstrated colorimetric LAMP detection using a modified commercial fluorescence-based isothermal LAMP reader, the BioRanger [25]. By removing the emission filters and monitoring scattering and reflection signals from the assays, the researchers were able to obtain real-time colorimetric curves.…”