PMC:7574920 / 2878-9446 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7574920","sourcedb":"PMC","sourceid":"7574920","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7574920","text":"INTRODUCTION\nThe coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus (1), is a major global health threat. A still unknown proportion of people, especially the elderly and those with preexisting conditions, are at high risk of a severe course of COVID-19 (2), leading to a high burden on health care systems worldwide. Further, because of limited testing capacity, only people with symptoms are usually tested for SARS-CoV-2 infection, although studies have confirmed that many individuals infected with SARS-CoV-2 are asymptomatic carriers of the virus (3, 4). This suggests that infection control strategies focusing on symptomatic patients are not sufficient to prevent virus spread.\nTherefore, large-scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively. This would allow for the rapid isolation of infected persons during an existing wave of infection. In addition, continuous and repeated testing of large groups within a population may be required as a long-term strategy to contain new outbreaks while keeping societies and economies functional until effective vaccines become available.\nAn active SARS-CoV-2 infection can be diagnosed by detecting either the viral genome or viral antigens in appropriate human samples. Assays for detecting SARS-CoV-2 antigens are limited by the sensitivity, specificity, and production speed of diagnostic antibodies, whereas detecting viral RNA only requires specific oligonucleotides. Therefore, an assay that detects SARS-CoV-2 RNA facilitates testing of large cohorts.\nThe SARS-CoV-2 diagnostic pipeline that has proven to be successful and that is currently used in many test centers consists of three steps: collecting nasopharyngeal or oropharyngeal swab specimens, isolation of total RNA, and specific detection of the viral genome by RT-qPCR. The latter comprises a reverse transcriptase (RT) step, which translates the viral RNA into DNA, followed by a semiquantitative DNA polymerase chain reaction using oligonucleotides specific for the viral cDNA (qPCR). As a result, a short piece of the viral genome is strongly amplified and then is detected by a sequence-specific oligonucleotide probe labeled with a fluorescent dye.\nThis procedure includes several steps that require sample handling; therefore, the detection process in a clinical diagnostic laboratory takes about 3 to 24 hours or more, depending on the number of samples and process optimization of the test center. In addition, in the context of the COVID-19 pandemic, many of the reagents required are only slowly being replenished due to insufficient production capacity or lack of international transport. Therefore, increasing daily test capacities for RT-qPCR–based diagnostics for SARS-CoV-2 RNA detection is currently limited. To accelerate and optimize such diagnostics, new scalable methods for RNA isolation and the detection of viral genomes are needed.\nAn alternative to RT-qPCR is reverse transcription loop-mediated isothermal amplification (RT-LAMP) (5–7). RT-LAMP reactions include a reverse transcriptase and a DNA polymerase with strong strand displacement activity and tolerance for elevated temperatures and up to six DNA oligonucleotides of a certain architecture. Samples with potential template molecules are added to the reaction and incubated for 20 to 60 min at a constant temperature (e.g., 65°C). The oligonucleotides act as primers for the reverse transcriptase, and additional oligonucleotides for the DNA polymerase are designed so the DNA products loop back at their ends. These, in turn, serve as self-priming templates for the DNA polymerase. In the presence of a few RNA template molecules, a chain reaction is set in motion, which then runs until the added reagents (in particular, the deoxynucleotide triphosphates) are used up.\nTo detect DNA production in RT-LAMP assays, various approaches have been described. One possibility is to use a pH indicator (e.g., phenol red) and run the reaction in a weakly buffered environment. As the chain reaction proceeds, the pH is lowered, which results in a visible color change from red to yellow making it an appealing assay for point-of-care diagnosis (8). Previously, RT-LAMP assays have been proposed for diagnostic detection of other RNA viruses, such as influenza virus (9). Also, several studies have demonstrated the use of isothermal DNA amplification to detect small amounts of SARS-CoV-2 RNA. The majority of these studies used in vitro transcribed (IVT) short fragments of the viral genomic RNA (10–12) and showed a detection limit of somewhere between 10 and 100 RNA molecules per reaction. For the detection of SARS-CoV-2 RNA, a few commercial rapid tests have been developed [reviewed in (13)] using isothermal DNA amplification reactions involving proprietary enzyme formulations that are not commercially available in a ready-to-go format. Further, their exact sensitivity is still subject to discussion owing to a lack of studies using sufficiently large numbers of test samples.\nThe performance of an RT-LAMP assay does not require expensive special equipment such as a thermal cycler with real-time fluorescence measurement, because positive samples are determined by a color change from red to yellow within 30 min after the start of the incubation at 65°C. For detection, simple mobile phone cameras, copy machines, office scanners, or plate scanners with spectrophotometric quantification can be used. During the early phase of the COVID-19 pandemic (early March 2020) in Germany, we tested the sensitivity and specificity of a colorimetric RT-LAMP assay for detecting SARS-CoV-2 RNA in clinical RNA samples isolated from pharyngeal swab specimens collected from individuals being tested for COVID-19 (and provided by the Heidelberg University Hospital’s diagnostic laboratory after removal of an aliquot for SARS-CoV-2 RNA testing by RT-qPCR) (fig. S1). We also developed a swab–to–RT-LAMP assay that used naso/oropharyngeal swab specimens directly without the need for an RNA isolation step. We tested \u003e700 clinical RNA samples with a wide range of viral loads, allowing us to determine accurately the sensitivity range of the colorimetric RT-LAMP assay. We also developed a multiplexed LAMP-sequencing protocol using barcoded Tn5 transposase tagmentation that enabled rapid identification of positive results in thousands of RT-LAMP reactions within the same next-generation sequencing 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