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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.
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BackgroundCurrently, more than 500 different AgPOCTs for SARS-CoV-2 diagnostics are on sale (July 2021), for many of which no data about sensitivity other than self-acclaimed values by the manufacturers are available. In many cases these do not reflect real-life diagnostic sensitivities. Therefore, manufacturer-independent quality checks of available AgPOCTs are needed, given the potential implications of false-negative results. ObjectiveThe objective of this study was to develop a scalable approach for direct comparison of the analytical sensitivities of commercially available SARS-CoV-2 antigen point-of-care tests (AgPOCTs) in order to rapidly identify poor performing products. MethodsWe present a methodology for quick assessment of the sensitivity of SARS-CoV-2 lateral flow test stripes suitable for quality evaluation of many different products. We established reference samples with high, medium and low SARS-CoV-2 viral loads along with a SARS-CoV-2 negative control sample. Test samples were used to semi-quantitatively assess the analytical sensitivities of 32 different commercial AgPOCTs in a head-to-head comparison. ResultsAmong 32 SARS-CoV-2 AgPOCTs tested, we observe sensitivity differences across a broad range of viral loads ([~]7.0*108 to [~]1.7*105 SARS-CoV-2 genome copies per ml). 23 AgPOCTs detected the Ct25 test sample ([~]1.4*106 copies/ ml), while only five tests detected the Ct28 test sample ([~]1.7*105 copies/ ml). In the low range of analytical sensitivity we found three saliva spit tests only delivering positive results for the Ct21 sample ([~]2.2*107 copies/ ml). Comparison with published data support our AgPOCT ranking. Importantly, we identified an AgPOCT offered in many local drugstores and supermarkets, which did not reliably recognize the sample with highest viral load (Ct16 test sample with [~]7.0*108 copies/ ml) leading to serious doubts in its usefulness in SARS-CoV-2 diagnostics. ConclusionThe rapid sensitivity assessment procedure presented here provides useful estimations on the analytical sensitivities of 32 AgPOCTs and identified a widely-spread AgPOCT with concerningly low sensitivity.
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McQ is a SARS-CoV-2 quantification assay that couples early-stage barcoding with high-throughput sequencing to enable multiplexed processing of thousands of samples. McQ is based on homemade enzymes to enable low-cost testing of large sample pools, circumventing supply chain shortages. Implementation of cost-efficient high-throughput methods for detection of RNA viruses such as SARS-CoV-2 is a potent strategy to curb ongoing and future pandemics. Here we describe Multiplexed SARS-CoV-2 Quantification platform (McQ), an in-expensive scalable framework for SARS-CoV-2 quantification in saliva samples. McQ is based on the parallel sequencing of barcoded amplicons generated from SARS- CoV-2 genomic RNA. McQ uses indexed, target-specific reverse transcription (RT) to generate barcoded cDNA for amplifying viral- and human-specific regions. The barcoding system enables early sample pooling to reduce hands-on time and makes the ap-proach scalable to thousands of samples per sequencing run. Robust and accurate quantification of viral load is achieved by measuring the abundance of Unique Molecular Identifiers (UMIs) introduced during reverse transcription. The use of homemade reverse transcriptase and polymerase enzymes and non-proprietary buffers reduces RNA to library reagent costs to 92 cents/sample and circumvents potential supply chain short-ages. We demonstrate the ability of McQ to robustly quantify various levels of viral RNA in 838 clinical samples and accu-rately diagnose positive and negative control samples in a test-ing workflow entailing self-sampling and automated RNA ex-traction from saliva. The implementation of McQ is modular, scalable and could be extended to other pathogenic targets in future.
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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.
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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.