Scalable RT-LAMP-based SARS-CoV-2 testing for infection surveillance with applications in pandemic control

Throughout the current SARS-CoV-2 pandemic, limited diagnostic testing capacity prevented sentinel testing of the population, demonstrating the need for novel testing strategies and infrastructures. Here, we describe the set-up of an alternative testing platform, which allows scalable surveillance testing as an acute pandemic response tool and for pandemic preparedness purposes, exemplified by SARS-CoV-2 diagnostics in an academic environment. The testing strategy involves self-sampling based on gargling saline, pseudonymized sample handling, automated 96-well plate-based RNA extraction, and viral RNA detection using a semi-quantitative multiplexed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay with an analytical sensitivity comparable to RT-quantitative polymerase chain reaction (RT-qPCR). We provide standard operating procedures and an integrated software solution for all workflows, including sample logistics, LAMP assay analysis by colorimetry or by sequencing (LAMP-seq), and communication of results to participants and the health authorities. Using large sample sets including longitudinal sample series we evaluated factors affecting the viral load and the stability of gargling samples as well as the diagnostic sensitivity of the RT-LAMP assay. We performed >35,000 tests during the pandemic, with an average turnover time of fewer than 6 hours from sample arrival at the test station to result announcement. Altogether, our work provides a blueprint for fast, sensitive, scalable, cost- and labor-efficient RT-LAMP diagnostics. As RT-LAMP-based testing requires advanced, but non-specialized laboratory equipment, it is independent of potentially limiting clinical diagnostics supply chains. One-sentence summaryA blueprint for scalable RT-LAMP test capacity for the sensitive detection of viral genomes demonstrated by SARS-CoV-2 surveillance testing.

SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and alternative, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric RT-LAMP using N primers, as well as RT-qPCR using E gene primers showing that the here presented RNA extraction protocol can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Screening for SARS-CoV-2 infections with colorimetric RT-LAMP and LAMP sequencing

The COVID-19 pandemic caused by the novel SARS-CoV-2 virus poses a significant public-health problem. In order to control the pandemic, rapid tests for detecting existing infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) hold outstanding promise towards greatly simplified and broadly applicable testing methods. RT-LAMP assays appear more robust than qPCR-based methods and only require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP protocol using clinical SARS-CoV-2 samples and also established a protocol that does not require prior RNA isolation ("swab-to-RT-LAMP"). Our study is based on several hundred clinical patient samples with a wide range of viral loads, thus allowing, for the first time, to accurately determine the sensitivity and specificity of the RT-LAMP assay for the detection of SARS-CoV-2 in patients. We found that RT-LAMP can reliably detect SARS-CoV-2 samples with a qPCR threshold cycle number (CT value) of up to 30 in the standard RT-qPCR assay. We used both, either purified RNA or direct pharyngeal swab specimens and showed that RT-LAMP assays have, despite a decreased sensitivity compared to RT-qPCR, excellent specificity. We also developed a multiplexed LAMP-sequencing protocol as a diagnostic and validation procedure to detect and record the outcome of RT-LAMP assays. LAMP-sequencing is fully scalable and can assess the results of thousands of LAMP reactions in parallel. Finally, we propose applications of RT-LAMP based assays for SARS-CoV-2 detection.