> top > projects > LitCovid-sentences > docs > PMC:7574920 > annotations
LitCovid-sentences  

PMC:7574920 JSONTXT 17 Projects

Annnotations TAB TSV DIC JSON TextAE

Id Subject Object Predicate Lexical cue
T1 0-97 Sentence denotes A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples
T2 99-248 Sentence denotes A colorimetric isothermal RNA amplification method was shown to detect SARS-CoV-2 RNA in clinical samples with excellent sensitivity and specificity.
T3 250-294 Sentence denotes A colorful test for SARS-CoV-2 RNA detection
T4 295-404 Sentence denotes We need simple methods to rapidly test large numbers of people for infection with the SARS-CoV-2 coronavirus.
T5 405-541 Sentence denotes Quantitative PCR (qPCR) after reverse transcription (RT), the standard method, is very sensitive but requires expensive instrumentation.
T6 542-658 Sentence denotes Loop-mediated isothermal amplification (LAMP) is an alternative to qPCR that is faster and requires fewer resources.
T7 659-823 Sentence denotes Dao Thi et al. tested the RT-LAMP assay on several hundred clinical RNA samples isolated from pharyngeal swabs collected from individuals being tested for COVID-19.
T8 824-968 Sentence denotes They confirmed that the RT-LAMP assay was a simpler albeit less sensitive option compared to RT-qPCR for large-scale testing for SARS-CoV-2 RNA.
T9 969-1212 Sentence denotes These investigators also developed a simplified version of this method (direct swab–to–RT-LAMP assay) that did not require a prior RNA isolation step as well as a method for highly multiplexed sequencing of RT-LAMP reactions (LAMP-sequencing).
T10 1214-1222 Sentence denotes Abstract
T11 1223-1394 Sentence denotes The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge.
T12 1395-1492 Sentence denotes Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical.
T13 1493-1679 Sentence denotes Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods.
T14 1680-1880 Sentence denotes Compared to RT quantitative polymerase chain reaction (RT-qPCR)–based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation.
T15 1881-2010 Sentence denotes Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene.
T16 2011-2159 Sentence denotes We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19.
T17 2160-2262 Sentence denotes We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA.
T18 2263-2499 Sentence denotes Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%.
T19 2500-2706 Sentence denotes We also developed a swab–to–RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay.
T20 2707-2876 Sentence denotes In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.
T21 2878-2890 Sentence denotes INTRODUCTION
T22 2891-3065 Sentence denotes The 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.
T23 3066-3277 Sentence denotes 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.
T24 3278-3520 Sentence denotes 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).
T25 3521-3645 Sentence denotes This suggests that infection control strategies focusing on symptomatic patients are not sufficient to prevent virus spread.
T26 3646-3793 Sentence denotes Therefore, large-scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively.
T27 3794-3892 Sentence denotes This would allow for the rapid isolation of infected persons during an existing wave of infection.
T28 3893-4130 Sentence denotes 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.
T29 4131-4263 Sentence denotes An active SARS-CoV-2 infection can be diagnosed by detecting either the viral genome or viral antigens in appropriate human samples.
T30 4264-4465 Sentence denotes 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.
T31 4466-4551 Sentence denotes Therefore, an assay that detects SARS-CoV-2 RNA facilitates testing of large cohorts.
T32 4552-4830 Sentence denotes The 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.
T33 4831-5047 Sentence denotes 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).
T34 5048-5214 Sentence denotes 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.
T35 5215-5466 Sentence denotes This 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.
T36 5467-5660 Sentence denotes 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.
T37 5661-5785 Sentence denotes Therefore, increasing daily test capacities for RT-qPCR–based diagnostics for SARS-CoV-2 RNA detection is currently limited.
T38 5786-5916 Sentence denotes To accelerate and optimize such diagnostics, new scalable methods for RNA isolation and the detection of viral genomes are needed.
T39 5917-6023 Sentence denotes An alternative to RT-qPCR is reverse transcription loop-mediated isothermal amplification (RT-LAMP) (5–7).
T40 6024-6237 Sentence denotes 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.
T41 6238-6376 Sentence denotes 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).
T42 6377-6556 Sentence denotes 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.
T43 6557-6628 Sentence denotes These, in turn, serve as self-priming templates for the DNA polymerase.
T44 6629-6817 Sentence denotes 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.
T45 6818-6901 Sentence denotes To detect DNA production in RT-LAMP assays, various approaches have been described.
T46 6902-7016 Sentence denotes One possibility is to use a pH indicator (e.g., phenol red) and run the reaction in a weakly buffered environment.
T47 7017-7188 Sentence denotes 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).
T48 7189-7310 Sentence denotes Previously, RT-LAMP assays have been proposed for diagnostic detection of other RNA viruses, such as influenza virus (9).
T49 7311-7433 Sentence denotes Also, several studies have demonstrated the use of isothermal DNA amplification to detect small amounts of SARS-CoV-2 RNA.
T50 7434-7633 Sentence denotes 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.
T51 7634-7886 Sentence denotes 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.
T52 7887-8027 Sentence denotes Further, their exact sensitivity is still subject to discussion owing to a lack of studies using sufficiently large numbers of test samples.
T53 8028-8308 Sentence denotes The 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.
T54 8309-8454 Sentence denotes For detection, simple mobile phone cameras, copy machines, office scanners, or plate scanners with spectrophotometric quantification can be used.
T55 8455-8907 Sentence denotes 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).
T56 8908-9046 Sentence denotes We also developed a swab–to–RT-LAMP assay that used naso/oropharyngeal swab specimens directly without the need for an RNA isolation step.
T57 9047-9209 Sentence denotes We tested >700 clinical RNA samples with a wide range of viral loads, allowing us to determine accurately the sensitivity range of the colorimetric RT-LAMP assay.
T58 9210-9446 Sentence denotes 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 run.
T59 9448-9455 Sentence denotes RESULTS
T60 9457-9552 Sentence denotes Establishing colorimetric RT-LAMP assay sensitivity using an artificial SARS-CoV-2 RNA template
T61 9553-9688 Sentence denotes To detect SARS-CoV-2 RNA with RT-LAMP, we used the WarmStart Colorimetric RT-LAMP 2X Master Mix (DNA and RNA) from New England Biolabs.
T62 9689-9807 Sentence denotes This mix contains two enzymes, an engineered reverse transcriptase (RTx) and a strand-displacing polymerase (Bst 2.0).
T63 9808-10059 Sentence denotes In addition, the reaction mixture contains oligonucleotide-based aptamers that function as reversible temperature-dependent inhibitors, ensuring that the reaction only runs at an elevated temperature (WarmStart) to avoid nonspecific priming reactions.
T64 10060-10272 Sentence denotes Several primer sets were recently proposed for RT-LAMP–based detection of SARS-CoV-2 RNA by Zhang et al. (11) and by Yu et al. (10), and these primer sets were subsequently validated with in vitro–translated RNA.
T65 10273-10469 Sentence denotes We prepared and tested two primer sets for different RNA sections of the SARS-CoV-2 genome, the N-A set targeting the N gene and the 1a-A set targeting open reading frame (ORF) 1a (table S1) (11).
T66 10470-10670 Sentence denotes Figure 1A shows that the oligonucleotide set for the N gene was capable of detecting 100 IVT RNA molecules in a test reaction with 1 μl of RNA solution, as evidenced by the red-to-yellow color change.
T67 10671-10723 Sentence denotes The reaction was conducted for up to 1 hour at 65°C.
T68 10724-10815 Sentence denotes For time points > 30 to 35 min, the negative control frequently became yellowish (Fig. 1A).
T69 10816-10916 Sentence denotes This was caused by spurious amplification products, which is a well-known problem with RT-LAMP (14).
T70 10917-11121 Sentence denotes Analysis by gel electrophoresis revealed clearly distinct banding patterns for the correct RT-LAMP reaction products (lanes with ≥100 molecules IVT RNA input) and the spurious reaction products (Fig. 1B).
T71 11122-11188 Sentence denotes Fig. 1 Sensitivity of the RT-LAMP assay determined using IVT RNA.
T72 11189-11335 Sentence denotes (A) Defined numbers of in vitro transcribed (IVT) RNA molecules of the SARS-CoV-2 N gene were added to the RT-LAMP reaction and incubated at 65°C.
T73 11336-11439 Sentence denotes At indicated times, samples were removed from the heating block and cooled on ice to stop the reaction.
T74 11440-11658 Sentence denotes Photographs were taken using the color scanner function of an office copy machine and show the red to yellow color change in positive samples. (B) The RT-LAMP reaction product (2.5 μl) was analyzed on a 2% agarose gel.
T75 11659-11869 Sentence denotes The typical band pattern of a successful RT-LAMP reaction was visible in the samples with 100 or more SARS-CoV-2 RNA molecules, i.e., in those samples that showed a color change from red to yellow after 30 min.
T76 11871-11935 Sentence denotes Testing clinical RNA samples with the colorimetric RT-LAMP assay
T77 11936-12063 Sentence denotes To evaluate the colorimetric RT-LAMP assay, we needed to compare its sensitivity and specificity to a validated RT-qPCR method.
T78 12064-12188 Sentence denotes We first used 95 RNA samples and performed RT-LAMP reactions using 1 μl of the isolated RNA in a reaction volume of 12.5 μl.
T79 12189-12320 Sentence denotes We detected a red-to-yellow color change in 36 of the samples following an incubation of the reaction for 30 min at 65°C (Fig. 2A).
T80 12321-12597 Sentence denotes To quantify the reaction, we used a plate scanner and measured the difference in absorbance (ΔOD) of the samples at 434 and 560 nm (corresponding to the absorbance maxima of the two forms of phenol red that were used in the assay as a pH-sensitive dye) at several time points.
T81 12598-12849 Sentence denotes To visualize the data, we plotted the ΔOD values against incubation time and colored the time traces of individual samples according to the cycle threshold (CT) values obtained from the RT-qPCR test run in the clinical diagnostic laboratory (Fig. 2B).
T82 12850-13110 Sentence denotes This RT-qPCR test was performed using a commercial diagnostic test kit containing a modified version of the E-Sarbeco primer set for the viral E gene suggested by Corman et al. (15) and 10 μl of RNA isolated with an automated platform (QiaSymphony or QiaCube).
T83 13111-13211 Sentence denotes Fig. 2 Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.
T84 13212-13324 Sentence denotes RNA samples isolated from 95 pharyngeal swab specimens were analyzed by the RT-LAMP assay using a 96-well plate.
T85 13325-13554 Sentence denotes The RT-LAMP reaction was incubated at 65°C, and the incubation was interrupted at different time points by cooling on ice for 30 s. (A) Photograph of the 96-well plate after a 30-min incubation at 65°C, taken with a mobile phone.
T86 13555-13797 Sentence denotes Wells with a yellow color indicate successful RT-LAMP amplification of a fragment of the SARS-CoV-2 N gene (using the N-A primer set). (B) Quantification of the red-to-yellow color change in all wells using spectrophotometric OD measurements.
T87 13798-13965 Sentence denotes The color value at the given time points is quantified as the difference between the wavelengths of the two absorbance maxima of phenol red: ΔOD = OD434 nm – OD560 nm.
T88 13966-14024 Sentence denotes Yellow (positive) samples yield a ΔOD of about 0.3 to 0.4.
T89 14025-14057 Sentence denotes Each line represents one sample.
T90 14058-14294 Sentence denotes For each sample, the line color indicates the CT (cycle threshold) value obtained from RT-qPCR data (using the E-Sarbeco primers) (15). (C) Scatter plot of ΔOD values at the 30-min time point from (B) compared to CT values from RT-qPCR.
T91 14295-14325 Sentence denotes Each dot is one sample (well).
T92 14326-14474 Sentence denotes In a colorimetric RT-LAMP reaction, positive samples with a CT < 30 changed the color of the phenol-red dye within the first 30 min of the reaction.
T93 14475-14652 Sentence denotes Samples with a CT > 30 either did not change their color or did so at time points > 35 min, simultaneously with a color change observed in some of the negative samples (Fig. 1).
T94 14653-14771 Sentence denotes On the basis of this observation, we used the ΔOD value at 30 min to decide whether a sample was positive or negative.
T95 14772-15063 Sentence denotes Plotting the ΔOD measurements versus CT values at the 30-min time point revealed that all patient samples with a CT < 30 showed a robust color change in the RT-LAMP test, whereas for samples with CT values between 30 and 35, a positive result was observed for only 1 of 10 samples (Fig. 2C).
T96 15064-15170 Sentence denotes This suggested a detection limit of the colorimetric RT-LAMP assay corresponding to a CT ≈ 30 for RT-qPCR.
T97 15171-15366 Sentence denotes The RT-qPCR kit used was calibrated and a CT ≈ 30 corresponded to 1000 RNA molecules present in the reaction according to the certificate provided by the manufacturer (see Materials and Methods).
T98 15367-15450 Sentence denotes The performance of each RT-qPCR run was validated using this as a positive control.
T99 15451-15691 Sentence denotes Considering that 10 μl of isolated RNA was used for RT-qPCR, but only 1 μl for the RT-LAMP assay, a cutoff of CT ≈ 30 agreed well with the observed experimental sensitivity of approximately 100 RNA molecules for the RT-LAMP assay (Fig. 1A).
T100 15692-15868 Sentence denotes Therefore, it appeared that the N-A primer set used for the RT-LAMP assay performed equally well with either IVT RNA or RNA samples isolated from the pharyngeal swab specimens.
T101 15869-16163 Sentence denotes In March 2020, at the beginning of the pandemic, the diagnostic laboratory that analyzed the pharyngeal swab samples by RT-qPCR validated all samples that tested positive with the E gene primer set in a second RT-qPCR using the N gene primer set, also of the Sarbeco sets of Corman et al. (15).
T102 16164-16311 Sentence denotes When plotting RT-LAMP assay results against the CT values for the N gene primer set, we observed a sensitivity cutoff of around CT ≈ 35 (fig. S2A).
T103 16312-16460 Sentence denotes Direct comparison of the CT values for the E gene and N gene primer sets for all samples revealed a difference of ~5.6 CT units (cycles) (fig. S2B).
T104 16461-16584 Sentence denotes This suggested that the N gene primers were less sensitive than the E gene primers for detecting SARS-CoV-2 RNA by RT-qPCR.
T105 16585-16723 Sentence denotes Similar differences have been observed previously for other primer sets, e.g., between the E gene primers and the RdRp-SARSr primers (16).
T106 16724-16991 Sentence denotes For the RT-LAMP assay, we also tested the 1a-A primer set directed against ORF1a (11) and found this primer set to be less sensitive than the N gene LAMP primer set, with a sensitivity cutoff of CT ≈ 25 when plotted against E gene RT-qPCR–derived CT values (fig. S3).
T107 16992-17162 Sentence denotes On the basis of these results, we decided to use the N-A primer set for the RT-LAMP assay and to compare our results with RT-qPCR performed with the E-Sarbeco primer set.
T108 17164-17237 Sentence denotes Validation of the colorimetric RT-LAMP assay for SARS-CoV-2 RNA detection
T109 17238-17346 Sentence denotes To determine the specificity and sensitivity of the RT-LAMP assay, we continued to analyze more RNA samples.
T110 17347-17422 Sentence denotes We assayed a total of 768 RNA samples obtained on different days (fig. S1).
T111 17423-17709 Sentence denotes Visualization of the RT-LAMP assay results 30 min after the start of the incubation at 65°C showed comparable behavior of the samples in a total of ten 96-well test plates (Fig. 3A and Table 1), indicating that the RT-LAMP assay was reproducible from day to day and from plate to plate.
T112 17710-17770 Sentence denotes Fig. 3 Detection of SARS-CoV-2 RNA using the RT-LAMP assay.
T113 17771-17896 Sentence denotes (A) Scatter plot shows a comparison of RT-LAMP assay results and RT-qPCR results for RNA samples tested on 10 96-well plates.
T114 17897-18011 Sentence denotes The RNA extraction method (QC, QiaCube, a column-based method; QS, QiaSymphony, a bead-based method) is indicated.
T115 18012-18127 Sentence denotes The time point for measurement by the colorimetric RT-LAMP assay was 30 min after the start of the 65°C incubation.
T116 18128-18183 Sentence denotes The 96-well plate shown in Fig. 2 is not included here.
T117 18184-18409 Sentence denotes Table 1 shows numbers of samples stratified according to the results of the RT-LAMP and the RT-qPCR assays. (B) Sensitivity (right) and specificity (left) of the RT-LAMP assay [derived from data in (A) and Table 1] are shown.
T118 18410-18524 Sentence denotes The specificity is the fraction of RT-qPCR–negative samples correctly identified as negative by the RT-LAMP assay.
T119 18525-18818 Sentence denotes For sensitivity, the RT-qPCR–positive samples were stratified by CT values into three bins (as indicated by x axis labels), and for each bin, the sensitivity is given as the fraction of qPCR-positive samples in the respective CT bin that have also given a positive result in the RT-LAMP assay.
T120 18819-19060 Sentence denotes The thick black lines indicate the values of these fractions (i.e., the specificity and sensitivity estimates); the black boxes indicate the corresponding 95% confidence intervals (Wilson’s binomial confidence interval). (See also table S2).
T121 19061-19175 Sentence denotes Table 1 Shown is RT-qPCR and RT-LAMP testing of 768 clinical samples stratified into CT value bins (see Fig. 3A).
T122 19176-19267 Sentence denotes Fig. 3B and table S2 show specificity and sensitivity values calculated from these numbers.
T123 19268-19275 Sentence denotes RT-LAMP
T124 19276-19295 Sentence denotes CT Pos Neg Sum
T125 19296-19327 Sentence denotes RT-qPCR Pos 0–25 51 0 51
T126 19328-19344 Sentence denotes 25–30 28 2 30
T127 19345-19361 Sentence denotes 30–35 4 16 20
T128 19362-19378 Sentence denotes 35–40 0 16 16
T129 19379-19401 Sentence denotes Neg Neg 2 649 651
T130 19402-19595 Sentence denotes Sum 85 683 768 The consistency of the results during the analysis confirmed a threshold of ΔOD > +0.3 as a robust measure to identify samples that were positive for SARS-CoV-2 RNA (Fig. 3A).
T131 19596-19795 Sentence denotes RT-qPCR–positive samples with a CT < 30 scored positive in the RT-LAMP assay (79 of 81), whereas almost all samples with CT values between 30 and 40 scored negative (only 4 positive of 36) (Fig. 3B).
T132 19796-19918 Sentence denotes This confirmed the sensitivity of the RT-LAMP assay for detection of SARS-CoV-2 RNA in samples corresponding to a CT < 30.
T133 19919-20077 Sentence denotes We observed small differences between different plates on the exact sensitivity threshold, probably caused by slight variability in plate or reagent handling.
T134 20078-20241 Sentence denotes We found two RT-qPCR–negative samples that scored positive in the RT-LAMP assay (Fig. 3A and Table 1) and one sample that scored just below the ΔOD cutoff of +0.3.
T135 20242-20330 Sentence denotes The overall specificity of the RT-LAMP test was 99.7% (Wilson’s 95% confidence interval:
T136 20331-20447 Sentence denotes 98.9 to 99.9%), and the sensitivity for samples with CT < 30 on RT-qPCR was 97.5% (Wilson’s 95% confidence interval:
T137 20448-20486 Sentence denotes 91.4 to 99.3%) (Fig. 3B and table S2).
T138 20488-20539 Sentence denotes Multiplexed sequencing of RT-LAMP reaction products
T139 20540-20689 Sentence denotes Our results indicated that the colorimetric RT-LAMP assay enabled robust identification of positive samples after a 25- to 30-min incubation at 65°C.
T140 20690-20823 Sentence denotes Validation of positive results, however, required confirmation that the RT-LAMP reaction led to the amplification of viral sequences.
T141 20824-20960 Sentence denotes To analyze the sequences of many RT-LAMP reaction products, we established multiplexed sequencing of RT-LAMP products (LAMP-sequencing).
T142 20961-21044 Sentence denotes LAMP-sequencing is based on Tn5 transposase tagmentation (17) and sample barcoding.
T143 21045-21166 Sentence denotes Tagmentation enables fragmentation and direct adapter ligation of DNA samples for analysis by next-generation sequencing.
T144 21167-21285 Sentence denotes We used a set of 96 barcoded adapters for tagmentation to barcode the RT-LAMP reaction products in each 96-well plate.
T145 21286-21422 Sentence denotes After tagmentation, all barcoded fragments from each plate were pooled and size-selected by bead purification to remove excess adapters.
T146 21423-21534 Sentence denotes A second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented RT-LAMP fragments.
T147 21535-21711 Sentence denotes Last, all amplified pools were combined for analysis using one next-generation sequencing run where the origin of each DNA fragment was specified by the two barcodes (Fig. 4A).
T148 21712-21790 Sentence denotes Fig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).
T149 21791-21833 Sentence denotes (A) Workflow for LAMP-sequencing is shown.
T150 21834-22012 Sentence denotes A plate of 96 barcoded (BC) adapters with unique molecular identifiers (UMIs) and mosaic ends (ME) was used as a seed plate for Tn5 tagmentation of all RT-LAMP reaction products.
T151 22013-22129 Sentence denotes After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection.
T152 22130-22475 Sentence denotes Each pool of tagmentation products was then amplified using primers with plate-specific barcodes, and the PCR products were analyzed by Illumina sequencing. (B) Comparison of the outcome of the three assays: LAMP-sequencing (purple, negative; green, positive; gray, too few UMIs), RT-LAMP (after 30-min incubation, y axis), and RT-qPCR (x axis).
T153 22476-22507 Sentence denotes Each dot represents one sample.
T154 22508-22667 Sentence denotes If a substantial number of the sequencing reads contained SARS-CoV-2 RNA, the sample was called positive (green), if not, then it was called negative (purple).
T155 22668-22970 Sentence denotes For some samples (gray), no LAMP-sequencing call could be made due to too few UMIs. (See also Table 2). (C) Although the RT-LAMP assay was scored after a 30-min incubation at 65°C (left), LAMP-sequencing was performed only after the samples had been incubated for another 10 min (15 min for one plate).
T156 22971-23152 Sentence denotes This panel shows the RT-LAMP assay outcome (y axis) scored after the full incubation time, whereas the RT-qPCR CT values (x axis) and LAMP-sequencing results are the same as in (B).
T157 23153-23356 Sentence denotes Of the LAMP-sequencing reads obtained, 98% mapped either to the part of the viral genome targeted by the RT-LAMP primers (80.6%) or contained short k-mers derived from primer sequences (17.4%) (fig. S4).
T158 23357-23426 Sentence denotes This indicated that LAMP-sequencing amplified the targeted sequences.
T159 23427-23593 Sentence denotes Reads containing only primer sequences were likely to be the result of spurious amplification products as these were also formed in the absence of input RNA (Fig. 1).
T160 23594-23892 Sentence denotes For quantification of individual LAMP reactions, we classified reads according to whether or not they contained viral sequences, which were not directly covered by the primers (orange segments in fig. S4A), and counted the reads for each sample (as specified by its barcode combination) (fig. S4B).
T161 23893-23971 Sentence denotes For 754 of the 768 samples, we obtained enough reads to make a call (fig. S5).
T162 23972-24148 Sentence denotes For the 754 samples that underwent successful LAMP-sequencing, the results confirmed all samples that scored positive on the RT-LAMP assay with a CT < 30 (Fig. 4B and Table 2).
T163 24149-24346 Sentence denotes For the two samples with a negative RT-qPCR result that scored positive on the RT-LAMP assay (Fig. 3), the LAMP-sequencing call agreed with the RT-qPCR result and thus corrected the RT-LAMP result.
T164 24347-24391 Sentence denotes Table 2 Summary of LAMP-sequencing results.
T165 24392-24646 Sentence denotes The cross tabulation of RT-qPCR and RT-LAMP assay results shown in Table 1 have been split into samples where sequencing of RT-LAMP reaction products (LAMP-sequencing) was positive (Pos), negative (Neg), or inconclusive (too few reads) (see also Fig. 4).
T166 24647-24654 Sentence denotes RT-LAMP
T167 24655-24674 Sentence denotes CT Pos Neg Sum
T168 24675-24732 Sentence denotes LAMP- sequencing Pos RT-qPCR Pos 0–25 49 0 49
T169 24733-24749 Sentence denotes 25–30 28 0 28
T170 24750-24764 Sentence denotes 30–35 4 0 4
T171 24765-24779 Sentence denotes 35–40 0 0 0
T172 24780-24798 Sentence denotes Neg Neg 0 0 0
T173 24799-24834 Sentence denotes Neg RT-qPCR Pos 0–25 0 0 0
T174 24835-24849 Sentence denotes 25–30 0 2 2
T175 24850-24866 Sentence denotes 30–35 0 16 16
T176 24867-24883 Sentence denotes 35–40 0 16 16
T177 24884-24906 Sentence denotes Neg Neg 2 637 639
T178 24907-24952 Sentence denotes Too few reads RT-qPCR Pos 0–25 2 0 2
T179 24953-24967 Sentence denotes 25–30 0 0 0
T180 24968-24982 Sentence denotes 30–35 0 0 0
T181 24983-24997 Sentence denotes 35–40 0 0 0
T182 24998-25018 Sentence denotes Neg Neg 0 12 12
T183 25019-25140 Sentence denotes Sum 85 683 768 LAMP-sequencing was performed using the RT-LAMP samples after a prolonged incubation of 40 min at 65°C.
T184 25141-25253 Sentence denotes At this time point, many of the negative samples and also samples with a CT between 30 and 40 had turned yellow.
T185 25254-25312 Sentence denotes LAMP-sequencing eliminated all of these samples (Fig. 4C).
T186 25313-25552 Sentence denotes This indicated that even for the RT-qPCR–positive samples with a CT between 30 and 35, the color change that took place at time points > 30 min was caused by spurious amplification products and not by late amplification of viral sequences.
T187 25553-25754 Sentence denotes These results therefore confirmed that LAMP-sequencing was able to assess the results of multiple RT-LAMP reactions in parallel and to identify false-positive samples in the colorimetric RT-LAMP assay.
T188 25756-25801 Sentence denotes A swab–to–RT-LAMP assay without RNA isolation
T189 25802-25917 Sentence denotes RNA isolation is time consuming, costly, and depends on reagents with potentially limited supply during a pandemic.
T190 25918-26131 Sentence denotes Alternative, noncommercial solutions for RNA isolation, e.g., using silica gel matrix or magnetic beads, require specialized knowledge and cannot be implemented easily for point-of-care or decentralized screening.
T191 26132-26350 Sentence denotes Several reports have indicated that RT-qPCR (18–20) and RT-LAMP assays (21, 22) are compatible with direct testing of nasopharyngeal and oropharyngeal swab specimens without a prior RNA purification or extraction step.
T192 26351-26543 Sentence denotes To establish an RT-LAMP assay that could test unprocessed specimens (swab–to–RT-LAMP assay), we first assessed the stability of naked RNA in swab specimens that were collected in Amies medium.
T193 26544-26676 Sentence denotes We titrated defined numbers of IVT RNA molecules of the SARS-CoV-2 N gene into swab samples from COVID-19–negative control subjects.
T194 26677-26873 Sentence denotes We tested different conditions, particularly the influence of detergent (to inactivate the virus) and heat (to denature the capsid and release the viral RNA as well as inactivate the virus) (figs.
T195 26874-26903 Sentence denotes S6 and S7, and data file S1).
T196 26904-27232 Sentence denotes Consistent with previous reports about other RNA viruses (23–25) and tests using heat inactivation of swab specimens for direct RT-qPCR assays (26), these experiments established that native swab specimens and heat-treated swab specimens were compatible for detection of SARS-CoV-2 RNA in swab samples from infected individuals.
T197 27234-27289 Sentence denotes Testing clinical samples with the swab–to–RT-LAMP assay
T198 27290-27507 Sentence denotes On the basis of these preliminary experiments, we decided to use swab samples either directly without any treatment (direct swab–to–RT-LAMP assay) or after heat treatment for 5 min at 95°C (hot swab–to–RT-LAMP assay).
T199 27508-27619 Sentence denotes As an additional precaution, we kept the samples in the cold (using an ice-cold metal block) whenever possible.
T200 27620-27727 Sentence denotes For testing large numbers of clinical samples, we performed the RT-LAMP assay using several 96-well plates.
T201 27728-27886 Sentence denotes In total, we tested 209 different samples using the hot swab–to–RT-LAMP assay, and of these, 131 samples also were tested by the direct swab–to–RT-LAMP assay.
T202 27887-28034 Sentence denotes Many samples were tested twice but using aliquots withdrawn at different time points (usually within 24 hours) from the swab samples stored at 4°C.
T203 28035-28155 Sentence denotes This resulted in 235 direct swab–to–RT-LAMP assay measurements and 343 hot swab–to–RT-LAMP assay measurements (Fig. 5A).
T204 28156-28398 Sentence denotes The hot swab–to–RT-LAMP assay detected a color change in the majority of samples with a CT < 30 with high sensitivity, whereas the direct swab–to–RT-LAMP assay only exhibited a high sensitivity for samples with a CT < 25 (Fig. 5 and Table 3).
T205 28399-28543 Sentence denotes The heat treatment rendered the RT-LAMP assay more stringent as it reduced false positives and more sensitive for samples with a CT of 25 to 30.
T206 28544-28648 Sentence denotes We found that some positive samples did not induce a color change but did so when assayed a second time.
T207 28649-28724 Sentence denotes We therefore would recommend running this assay using technical duplicates.
T208 28725-28791 Sentence denotes Fig. 5 Swab–to–RT-LAMP assay of clinical pharyngeal swab samples.
T209 28792-28965 Sentence denotes (A) Skipping a prior RNA isolation step, pharyngeal swab samples were subjected to the RT-LAMP assay either directly (left) or after 5 min of heat treatment at 95°C (right).
T210 28966-29104 Sentence denotes For each sample, scatter plots are used to compare the swab–to–RT-LAMP assay results (ΔOD values) with the results of RT-qPCR (CT values).
T211 29105-29377 Sentence denotes The measurement time point was 30 min after the start of the 65°C incubation. (B) Shown is the sensitivity (right) and specificity (left) of the swab–to–RT-LAMP assay [derived from the data in (A)] using the decision threshold indicated by the horizontal gray line in (A).
T212 29378-29619 Sentence denotes Specificity and sensitivity values (thick lines) are shown with their 95% confidence intervals (boxes) as in Fig. 3, with blue indicating the direct swab–to–RT-LAMP assay and red indicating the hot swab–to–RT-LAMP assay. (Also see table S3).
T213 29620-29734 Sentence denotes Table 3 Shown is RT-qPCR and RT-LAMP testing of 592 clinical samples stratified into CT value bins (see Fig. 5A).
T214 29735-29826 Sentence denotes Fig. 5A and table S3 show specificity and sensitivity values calculated from these numbers.
T215 29827-29857 Sentence denotes Hot swab–to–RT-LAMP RT-LAMP
T216 29858-29878 Sentence denotes CT Pos Neg Sum
T217 29879-29910 Sentence denotes RT-qPCR Pos 0–25 38 4 42
T218 29911-29927 Sentence denotes 25–30 17 5 22
T219 29928-29944 Sentence denotes 30–35 5 23 28
T220 29945-29961 Sentence denotes 35–40 0 36 36
T221 29962-29984 Sentence denotes Neg Neg 1 214 215
T222 29985-30002 Sentence denotes Sum 61 282 343
T223 30003-30036 Sentence denotes Direct swab–to–RT-LAMP RT-LAMP
T224 30037-30057 Sentence denotes CT Pos Neg Sum
T225 30058-30089 Sentence denotes RT-qPCR Pos 0–25 15 1 16
T226 30090-30106 Sentence denotes 25–30 6 11 17
T227 30107-30123 Sentence denotes 30–35 2 21 23
T228 30124-30140 Sentence denotes 35–40 3 23 26
T229 30141-30163 Sentence denotes Neg Neg 9 144 153
T230 30164-30181 Sentence denotes Sum 35 200 235
T231 30183-30240 Sentence denotes Heterogeneity of specimen pH in the swab–to–RT-LAMP assay
T232 30241-30451 Sentence denotes Comparison of the results of the direct swab–to–RT-LAMP assay with the RT-LAMP assay using isolated RNA revealed a much broader distribution of the ΔOD measurements in negative samples (Fig. 5A versus Fig. 3A).
T233 30452-30558 Sentence denotes This was likely due to a sample-specific variability that influenced the starting pH in the LAMP reaction.
T234 30559-30645 Sentence denotes This might have affected the interpretability of the measurement at 30 min (ΔOD30min).
T235 30646-30709 Sentence denotes We investigated how this pH shift influenced the RT-LAMP assay.
T236 30710-30842 Sentence denotes For three plates, the data acquired for the RT-LAMP assay also included measurements for the 10-min time point (ΔOD10min) (Fig. 6A).
T237 30843-31025 Sentence denotes We plotted the change of the ΔOD between the 10- and 30-min time points (i.e., the difference ΔOD30min – ΔOD10min, corresponding to the slope of the lines) versus ΔOD30min (Fig. 6B).
T238 31026-31208 Sentence denotes This removed the variability of the values for samples that did not change their color (negative samples) and permitted a better separation of the positive from the negative samples.
T239 31209-31278 Sentence denotes Fig. 6 Colorimetric readouts of the swab–to–RT-LAMP assay over time.
T240 31279-31403 Sentence denotes (A) The colorimetric readouts (ΔOD) for the direct (left) and hot (right) swab–to–RT-LAMP assays were assessed every 10 min.
T241 31404-31541 Sentence denotes Heterogeneity is notable at the early time points. ΔOD values at the zero time point were not measured for the hot swab–to–RT-LAMP assay.
T242 31542-31602 Sentence denotes Also, the 40-min time point was not available for one plate.
T243 31603-31728 Sentence denotes The kink in some lines at 30 min (right) was due to a transient equipment malfunction. (B) Comparison of two scoring schemes.
T244 31729-32030 Sentence denotes The readout used in Fig. 5 to score the direct (left) and hot (right) swab–to–RT-LAMP assays, namely, ΔOD at 30 min, is shown on the y axis, and compared to an alternative score, namely, the difference between the ΔOD signals at 30 min and at 10 min after the start of incubation, shown on the x axis.
T245 32031-32104 Sentence denotes The latter shows better separation between positive and negative samples.
T246 32105-32250 Sentence denotes We noticed that the pH variability depended on the sample volume used for the RT-LAMP assay and the composition of the medium used for the swabs.
T247 32251-32416 Sentence denotes For swabs in Amies medium (which was used for the clinical samples in this study), an RT-LAMP assay containing 1 μl of sample in a total volume of 20 μl was optimal.
T248 32417-32589 Sentence denotes Our results obtained using native and heat-treated swab specimens suggested better performance when using heat treatment of swab specimens before running the RT-LAMP assay.
T249 32591-32601 Sentence denotes DISCUSSION
T250 32602-32708 Sentence denotes Here, we evaluated the use and suitability of the RT-LAMP assay for the detection of SARS-CoV-2 infection.
T251 32709-32847 Sentence denotes We also developed LAMP-sequencing as a fully scalable alternative to colorimetric or fluorometric analysis of DNA amplification reactions.
T252 32848-33059 Sentence denotes Our results indicate that whereas the RT-LAMP assay using the N-A primer set is not sensitive enough to replace RT-qPCR in all applications, it does hold promise as a method for testing large numbers of samples.
T253 33060-33223 Sentence denotes We tested the RT-LAMP primer sets suggested by Zhang et al. (11) and found that the N-A primer set for the N gene worked better than the 1a-A primer set for ORF1a.
T254 33224-33492 Sentence denotes For samples with a CT ≤ 30 as measured by RT-qPCR with E-Sarbeco primers, we found overall satisfactory sensitivity and specificity values for SARS-CoV-2 RNA detection by the RT-LAMP assay using RNA samples isolated from pharyngeal swab specimens (Fig. 3 and Table 1).
T255 33493-33561 Sentence denotes For samples with CT > 30, the RT-LAMP assay was much less sensitive.
T256 33562-33679 Sentence denotes However, there is debate about which CT value for a positive RT-qPCR result should be considered clinically relevant.
T257 33680-33778 Sentence denotes Vogels et al. (16) indicate that a CT value above 36 corresponds to less than 10 molecules of RNA.
T258 33779-33945 Sentence denotes On the basis of our data, we conclude that the colorimetric RT-LAMP assay would be suitable for identifying individuals with a high or moderate SARS-CoV-2 viral load.
T259 33946-34205 Sentence denotes On the other hand, for those with a low viral load (at the onset of illness or during later stages of the disease), the sensitivity of the RT-LAMP assay, in its current implementation using the N-A primer set, is insufficient to detect a SARS-CoV-2 infection.
T260 34206-34467 Sentence denotes A number of other LAMP primer sets have been proposed and initially tested (21, 27, 28), showing that optimized primers and the use of combinations of primer sets hold promise to further increase the sensitivity of the RT-LAMP assay for detecting viral genomes.
T261 34468-34558 Sentence denotes Furthermore, alternative sample types, e.g., sputum or stool (29), might be more reliable.
T262 34559-34785 Sentence denotes One promising lead for future applications is the exploration of the hot swab–to–RT-LAMP assay using saliva specimens, although the relative sensitivity compared to using pharyngeal swab specimens is currently unclear (30–33).
T263 34786-34901 Sentence denotes Compatibility of the RT-LAMP assay with direct saliva specimens has been shown using spike-in experiments (22, 34).
T264 34902-35049 Sentence denotes Although faster and more convenient, the direct swab–to–RT-LAMP assay was less sensitive and less robust than the RT-LAMP assay using isolated RNA.
T265 35050-35312 Sentence denotes To increase robustness, various treatments of crude swab samples have been described previously [reviewed in (35)], many of which require additional processing of the samples, for example, by pipetting or by adding proteinase K to degrade contaminating proteins.
T266 35313-35530 Sentence denotes Rabe and Cepko (22) have suggested using cheap silica preparations and new sample inactivation protocols to enrich the RNA before the RT-LAMP assay, but this would complicate the simple swab–to–RT-LAMP assay workflow.
T267 35531-35786 Sentence denotes Last, our analysis found that a short heat treatment of 5 min at 95°C, which poses minimal additional handling steps, did not destroy the RNA but rather stabilized it and this improved the sensitivity and specificity of the swab–to–RT-LAMP assay (Fig. 5).
T268 35787-35932 Sentence denotes The heat likely helped to homogenize the sample, to inactivate ribonucleases (RNAses), and to break up the viral capsid to release the viral RNA.
T269 35933-36130 Sentence denotes Overall, our data demonstrate the feasibility of using a swab–to–RT-LAMP test and suggest applications especially in scenarios where RNA isolation is not available, e.g., in resource-poor settings.
T270 36131-36310 Sentence denotes In such cases, the hot swab–to–RT-LAMP assay seems a good option given that the direct swab–to–RT-LAMP assay yields a number of false positives due to spurious amplification (14).
T271 36311-36515 Sentence denotes Although spike-in experiments with IVT RNA can be informative, we have experienced clear differences when comparing such experiments to those using clinical RNA samples isolated from swab specimens (figs.
T272 36516-36545 Sentence denotes S6 and S7, and data file S1).
T273 36546-36721 Sentence denotes We therefore recommend validating any new proposed rapid SARS-CoV-2 diagnostic test using “real-life” clinical samples including a large fraction of negative clinical samples.
T274 36722-36906 Sentence denotes To overcome the problem of spurious amplification, an expanded oligonucleotide set that incorporates sequence-specific probes (34) or a CRISPR/Cas12a–based approach (36) could be used.
T275 36907-37004 Sentence denotes However, these applications have yet to be tested with large numbers of diverse clinical samples.
T276 37005-37073 Sentence denotes There are several differences between the RT-LAMP assay and RT-qPCR.
T277 37074-37303 Sentence denotes First, RT-qPCR requires a thermocycler to conduct the DNA amplification reaction, which is an expensive instrument, whereas isothermal incubation of RT-LAMP reactions can be conducted using a simple water bath or a heating block.
T278 37304-37378 Sentence denotes This makes the RT-LAMP assay more amenable for point-of-care applications.
T279 37379-37496 Sentence denotes Second, the reagents for the RT-LAMP assay are different from the ones used for RT-qPCR and are supplier independent.
T280 37497-37667 Sentence denotes According to the supplier of the RT-LAMP reagents used in this study (New England Biolabs), production of RT-LAMP reagents can be easily ramped up to satisfy high demand.
T281 37668-37842 Sentence denotes Third, the RT-LAMP assay, when combined with LAMP-sequencing, is suitable for analyzing large numbers of RT-LAMP reactions owing to the fully scalable DNA barcoding strategy.
T282 37843-37980 Sentence denotes In contrast, there are several hurdles to scaling up RT-qPCR assays, the major hurdle being the need for a large number of thermocyclers.
T283 37981-38084 Sentence denotes The RT-LAMP assay overcomes this problem and therefore will be a more scalable method for mass testing.
T284 38086-38151 Sentence denotes Application of RT-LAMP and LAMP-sequencing for SARS-CoV-2 testing
T285 38152-38259 Sentence denotes With its good sensitivity for samples up to CT ≈ 30, the colorimetric RT-LAMP assay has several advantages:
T286 38260-38331 Sentence denotes It is fast, inexpensive, and it can be evaluated without any equipment.
T287 38332-38580 Sentence denotes RT-LAMP reactions also appear to be less sensitive to contaminants in the samples than RT-qPCR, but care has to be taken that the samples used do not alter the pH as the colorimetric RT-LAMP assay is performed under conditions of weak pH buffering.
T288 38581-38745 Sentence denotes Some clinical samples contain contaminants that can lead to acidification of the reaction independent of the presence of a template RNA if too much sample is added.
T289 38746-39023 Sentence denotes Diagnostic RT-qPCR tests usually include a technical internal control, i.e., another RNA species, which is spiked into all samples and which is detected independent of the gene of interest to safeguard against the possibility of a general reaction failure within a sample tube.
T290 39024-39097 Sentence denotes It would be desirable to have a similar precaution for the RT-LAMP assay.
T291 39098-39226 Sentence denotes A multiplexed fluorescence readout might provide this (34) but comes at the expense of the simplicity of a colorimetric readout.
T292 39227-39521 Sentence denotes Our particular implementation of deep sequencing to analyze many RT-LAMP reactions simultaneously uses two sets of barcoded primers and is fully scalable so that, in one sequencing run, many thousands of LAMP reactions can be quantitatively analyzed for the presence of viral genomic sequences.
T293 39522-39708 Sentence denotes Although we used Illumina dye sequencing, more scalable sequencing technologies, such as Oxford Nanopore Technologies sequencing, could be used for amplicon sequencing and counting (37).
T294 39709-39853 Sentence denotes The workflow shown here uses LAMP-sequencing as a validation and backup procedure to double check the results of the colorimetric RT-LAMP assay.
T295 39854-40089 Sentence denotes However, LAMP-sequencing could also facilitate scale-up of the workflow for direct analysis of many thousands of samples in an efficient manner, provided that an infrastructure is established that allows the collection of such samples.
T296 40090-40197 Sentence denotes Thus, LAMP-sequencing could become an important part of workflows for routine testing of large populations.
T297 40198-40409 Sentence denotes Schmid-Burgk et al. (38) proposed decentralized RT-LAMP assays using combinatorial primer barcoding and centralized mass analysis of RT-LAMP products by next-generation sequencing as a means to scale-up testing.
T298 40410-40635 Sentence denotes Although this poses additional challenges in generating the individualized RT-LAMP assay reagents, it would simplify sample handling on the analytical side and it can be easily combined with the barcoding strategy shown here.
T299 40636-40679 Sentence denotes There are several limitations to our study.
T300 40680-40790 Sentence denotes We used surplus RNA sample material from a diagnostic laboratory rather than newly collected clinical samples.
T301 40791-40894 Sentence denotes The criteria for testing individuals may have influenced cohort characteristics and hence our findings.
T302 40895-41066 Sentence denotes It is not clear yet how well viral load as indicated by CT values from RT-qPCR assays informs about the degree of infectivity of an individual with a SARS-CoV-2 infection.
T303 41067-41278 Sentence denotes Therefore, we cannot say how our findings on the sensitivity of the RT-LAMP assay in comparison to RT-qPCR would translate into sensitivity for detecting infectious individuals who are shedding SARS-CoV-2 virus.
T304 41279-41479 Sentence denotes Moreover, the measured viral load does not indicate the course of a SARS-CoV-2 infection, as even individuals with a very low measured viral load can still develop severe symptoms of COVID-19 disease.
T305 41480-41679 Sentence denotes This may be, in part, because the viral load in a clinical sample taken from a specific site such as the pharynx is not representative of the overall viral burden that an infected individual carries.
T306 41680-41782 Sentence denotes We used LAMP-sequencing to validate the RT-LAMP assay results and did not use it as a diagnostic tool.
T307 41783-41960 Sentence denotes LAMP-sequencing is dependent on the sensitivity of the RT-LAMP reaction as it cannot detect false negative results caused by a failure of the RT-LAMP assay to amplify viral RNA.
T308 41961-42082 Sentence denotes Also, reagents such as the primer sets for the RT-LAMP assay may be subject to production-dependent quality fluctuations.
T309 42083-42196 Sentence denotes Therefore, all reagents must be precisely validated (batch control) before using an RT-LAMP assay diagnostically.
T310 42197-42311 Sentence denotes Application of the RT-LAMP assay has great potential, even more so as more sensitive primer sets become available.
T311 42312-42452 Sentence denotes The RT-LAMP assay and LAMP-sequencing could offer scalable testing that would be difficult to achieve with conventional RT-qPCR–based tests.
T312 42453-42651 Sentence denotes For example, the RT-LAMP assay could be used for regular testing of a whole workforce or in sentinel testing, ideally combined with simplified sample collection, e.g., in the form of saliva samples.
T313 42652-42876 Sentence denotes The RT-LAMP assay and LAMP-sequencing extend the range of available test methods and complement individual tests and pooled tests based on RT-qPCR (39) with a faster, simpler, and potentially more cost-effective test method.
T314 42878-42899 Sentence denotes MATERIALS AND METHODS
T315 42901-42913 Sentence denotes Study design
T316 42914-43099 Sentence denotes The intent of this study was to develop a clinical method for detecting SARS-CoV-2 RNA in RNA samples isolated from pharyngeal swab specimens from individuals being tested for COVID-19.
T317 43100-43311 Sentence denotes We used pseudo-anonymized surplus RNA sample material that had been collected for clinical diagnosis of SARS-CoV-2 infection by RT-qPCR carried out by the diagnostic laboratory of Heidelberg University Hospital.
T318 43312-43518 Sentence denotes Such reuse of material is in accordance with German regulations, which allow development and improvement of diagnostic assays using patient samples collected specifically to perform the testing in question.
T319 43519-43693 Sentence denotes Pharyngeal swab specimens provided to us were either collected through the nose (nasopharyngeal) or the mouth (oropharyngeal), or sometimes one swab was used to collect both.
T320 43694-43947 Sentence denotes Our study was designed to investigate the sensitivity and specificity of a colorimetric RT-LAMP assay and to evaluate its suitability as an alternative to RT-qPCR testing for detecting SARS-CoV-2 viral RNA in RNA isolated from pharyngeal swab specimens.
T321 43948-44023 Sentence denotes This study was conducted in Heidelberg, Germany in March and April of 2020.
T322 44024-44302 Sentence denotes The study was designed to first evaluate different existing primer sets for RT-LAMP reactions and to use them for (i) detection of SARS-CoV-2 RNA in RNA isolated from pharyngeal swabs and (ii) detection of SARS-CoV-2 RNA directly from swab specimens without prior RNA isolation.
T323 44303-44577 Sentence denotes All RNA samples used were pseudo-anonymized surplus material from the Heidelberg University Hospital diagnostic laboratory, and RT-qPCR results for these RNA samples were retrieved from the laboratory’s database only after the samples had been analyzed by the RT-LAMP assay.
T324 44578-44727 Sentence denotes The study design was to conduct RT-LAMP testing until sufficient samples (at least several hundreds) had been analyzed to obtain a conclusive result.
T325 44728-44907 Sentence denotes We also designed a deep sequencing-based method to validate the outcome of the RT-LAMP reactions using a Tn5 transposase–based fully scalable barcoding strategy (LAMP-sequencing).
T326 44909-44933 Sentence denotes Clinical sample handling
T327 44934-45047 Sentence denotes Specimens were collected as nasopharyngeal and oropharyngeal flocked swabs in Amies medium (eSwab, Copan Italia).
T328 45048-45220 Sentence denotes The sample collection happened as part of the routine operation of Heidelberg University Hospital and at public testing stations set up by the City of Heidelberg (fig. S1).
T329 45221-45407 Sentence denotes Collected samples were transported in sterile containers, delivered to the diagnostic laboratory within a few hours, and then examined directly or stored at 4°C until further processing.
T330 45408-45519 Sentence denotes Samples were processed in a biosafety level 2 cabinet until inactivation by heat or mixing with a lysis buffer.
T331 45521-45546 Sentence denotes RNA isolation and RT-qPCR
T332 45547-45894 Sentence denotes The standard diagnostic pipeline of the hospital laboratory was as follows: RNA was isolated from nasopharyngeal and oropharyngeal swab specimens using QIAGEN kits (QIAGEN, Hilden, Germany); either automated on the QIASymphony (DSP Virus/Pathogen Mini Kits) or QIAcube (QIAamp Viral RNA Mini Kits) devices or manually (QIAamp Viral RNA Mini Kits).
T333 45895-46075 Sentence denotes Please note that the QiaCube uses a sample volume of 140 μl and an elution volume of 100 μl, whereas the QiaSymphony uses a sample volume of 200 μl and an elution volume of 115 μl.
T334 46076-46223 Sentence denotes RT-qPCR for the quantification of the SARS-CoV-2 viral genome was performed using kits and reagents from TIB MOLBIO Syntheselabor, Berlin, Germany.
T335 46224-46380 Sentence denotes The kits were used according to the manufacturer’s instruction and contained the primer/probe sets developed based on the published Sarbeco primer set (15).
T336 46381-46594 Sentence denotes Per 20-μl reaction, the master mix contained 5.4 μl of RNAse free water, 4.0 μl of LightCycler Multiplex RNA Virus Master (Roche, Basel, Switzerland), 0.5 μl of LightMix Modular SARS and Wuhan CoV E gene (cat. no.
T337 46595-46711 Sentence denotes 53-0776-96; TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) or LightMix Modular SARS and Wuhan CoV N gene (cat. no.
T338 46712-46801 Sentence denotes 53-0775-96; TIB MOLBIOL), 0.5 μl of LightMix Modular EAV RNA Extraction Control (cat. no.
T339 46802-46939 Sentence denotes 66-0909-96; TIB MOLBIOL), and 0.1 μl of reverse transcriptase enzyme (LightCycler Multiplex RNA Virus Master, Roche, Basel, Switzerland).
T340 46940-47058 Sentence denotes The master mix (10 μl) was distributed per reaction into 96-well plates, and 10 μl of purified RNA was added per well.
T341 47059-47144 Sentence denotes The performance of the RT-qPCR was validated using a positive control for the E gene.
T342 47145-47229 Sentence denotes A total of 103 molecules of E gene RNA per RT-qPCR reaction correspond to a CT ≈ 30.
T343 47231-47273 Sentence denotes RT-LAMP primer design and positive control
T344 47274-47489 Sentence denotes The RT-LAMP primer sets used in this study have been designed by Zhang et al. (11) against ORF1a and N gene and were synthesized by Sigma-Aldrich (synthesis scale, 0.025 μmol; purification, desalt; solution, water).
T345 47490-47625 Sentence denotes The sequences and the concentrations of each oligonucleotide in the 10× primer mix used for the RT-LAMP assay can be found in table S1.
T346 47626-47999 Sentence denotes An RNA-positive control for the N gene was amplified from a short fragment from 2019-nCoV_N_Positive control plasmid [Integrated DNA Technologies (IDT), 10006625] with oligonucleotides T7-GeneN-Fragment.for and GeneN-Fragment.rev including the T7 promoter and a subsequent IVT with the MEGAscript T7 Kit (Invitrogen) purified using the RNeasy MinElute Cleanup Kit (QIAGEN).
T347 48001-48084 Sentence denotes Liquid handling using 96-well plates and precautions taken to prevent contamination
T348 48085-48151 Sentence denotes To prevent cross-contamination, we have taken several precautions.
T349 48152-48258 Sentence denotes The 10× primer mix was prepared with nuclease-free water (AM9937, Ambion) and stored in aliquots at −20°C.
T350 48259-48427 Sentence denotes To set up an RT-LAMP test, the RT-LAMP master mix was prepared freshly immediately before the test at a separate workspace with a dedicated pipette set and filter tips.
T351 48428-48548 Sentence denotes The 96-well PCR plate containing the RT-LAMP mix was covered with an Society for Biomolecular Screening (SBS) plate lid.
T352 48549-48694 Sentence denotes To avoid mix-ups during sample addition through well-by-well pipetting, the RNA or swab specimens were first collected into a 96-well seed plate.
T353 48695-48866 Sentence denotes The RNA was then added to the plate with the LAMP reagents at a dedicated workspace with a manual 96-channel pipettor (Liquidator 20 μl, Mettler Toledo) using filter tips.
T354 48867-49086 Sentence denotes The RT-LAMP and the RNA seed plate were instantly sealed with an optically clear adhesive seal (GK480-OS, Kisker Biotech) and an adhesive aluminum foil seal (SL-AM0550, Steinbrenner Laborsysteme, Germany), respectively.
T355 49087-49301 Sentence denotes If the product of an RT-LAMP reaction had to be analyzed by gel electrophoresis, the plate was opened with extreme caution at a separated post-LAMP workspace and loaded onto an agarose gel with a dedicated pipette.
T356 49303-49316 Sentence denotes RT-LAMP assay
T357 49317-49458 Sentence denotes Assays were assembled in total reaction volumes of either 12.5 μl (for LAMP assays using isolated RNA) or 20 μl (for swab–to–RT-LAMP assays).
T358 49459-49793 Sentence denotes Master mixes were prepared at room temperature for each reaction immediately before use with either 6.25 or 10 μl, respectively, of the WarmStart Colorimetric RT-LAMP 2X Master Mix (M1800, New England Biolabs) and 1.25 or 2 μl, respectively, of the 10× primer mix, filled up to 11.5 or 19 μl with nuclease-free water (AM9937, Ambion).
T359 49794-49828 Sentence denotes Values given are for one reaction:
T360 49829-50108 Sentence denotes For a 96-well plate, 100 times larger volumes were used, and the LAMP mix was distributed to the wells of a 96-well plate (4ti-0960/C, Brooks Life Sciences or 0030128672, Eppendorf) before pipetting 1 μl of sample into each well of the plate; for details, see previous paragraph.
T361 50109-50290 Sentence denotes Plates were prepared immediately before use to limit exposure of the LAMP reagents to atmospheric CO2 (to prevent acidification of the reaction) and kept on an ice-cold metal block.
T362 50291-50474 Sentence denotes Plates were sealed using a transparent adhesive foil (GK480-OS, Kisker Biotech), and the reactions were incubated in a PCR cycler at 65°C for 15 to 60 min with the lid heated to 75°C.
T363 50475-50625 Sentence denotes To perform measurements at the indicated time points, the reactions were taken out of the PCR cycler and placed into an ice cold metal block for 30 s.
T364 50626-50676 Sentence denotes This intensifies the color before the measurement.
T365 50677-50777 Sentence denotes Photographs were taken with cell phone cameras or the scanner function of an office copying machine.
T366 50779-50817 Sentence denotes Quantification of the RT-LAMP reaction
T367 50818-50934 Sentence denotes Absorbance measurements were performed with a Spark Cyto or Infinite M200 (Tecan) at 434 and 560 nm with 25 flashes.
T368 50935-51095 Sentence denotes These two peaks from phenol red are strongly changing during the acidification of the reaction (434 nm absorbance is increased, 560 nm absorbance is decreased).
T369 51096-51202 Sentence denotes To obtain a good readout of the color change, absorbance at 560 nm was substracted from the one at 434 nm.
T370 51203-51235 Sentence denotes This difference was denoted ΔOD.
T371 51237-51258 Sentence denotes Swab–to–RT-LAMP assay
T372 51259-51374 Sentence denotes For direct and hot swab–to–RT-LAMP assays, patient swab specimens were transferred first onto a 96-well seed plate.
T373 51375-51535 Sentence denotes For the direct assay, we then transferred 1 μl of the specimen directly to 19 μl of LAMP mix per well in a ready-made 96-well PCR plate (0030128672, Eppendorf).
T374 51536-51654 Sentence denotes The plate was sealed using a transparent adhesive foil (GK480-OS, Kisker Biotech) and kept on an ice-cold metal block.
T375 51655-51836 Sentence denotes For the hot assay, we sealed the seed plate with a pierceable lid (4ti-0566/96, Brooks Life Sciences) and heated it in a PCR cycler for 5 min at 95°C (with the lid heated to 105°C).
T376 51837-51902 Sentence denotes The seed plate was cooled down to 4°C on an ice-cold metal block.
T377 51903-52036 Sentence denotes Afterward, 1 μl of the heat-treated patient specimens was quickly added to a second ready-made plate with 19 μl of LAMP mix per well.
T378 52037-52122 Sentence denotes This plate was also sealed with transparent adhesive foil (GK480-OS, Kisker Biotech).
T379 52123-52196 Sentence denotes Both plates were then incubated at 65°C for the LAMP reaction to proceed.
T380 52197-52358 Sentence denotes For both swab–to–RT-LAMP assays, the PCR plates were briefly spun down and then incubated in a PCR cycler at 65°C for 10 to 60 min (with the lid heated to 75°C).
T381 52359-52509 Sentence denotes To perform measurements at the indicated time points, the reactions were taken out of the PCR cycler and placed into an ice-cold metal block for 30 s.
T382 52511-52533 Sentence denotes LAMP-sequencing method
T383 52534-52754 Sentence denotes Sequencing libraries for detecting viral sequences in RT-LAMP products were prepared by a modified Anchor-Seq protocol (37, 40) using Tn5 transposase tagmentation instead of sonication for genomic DNA fragmentation (17).
T384 52755-52803 Sentence denotes The relevant primers are summarized in table S4.
T385 52804-53111 Sentence denotes In detail, transposon adapters containing well-defining barcodes and unique molecular identifiers (UMIs) were annealed by mixing 25 μM oligos (P5-UMI-xi5001…5096-ME.fw, Tn5hY-Rd2-Wat-SC3) in 5 μM tris-HCl (pH 8), incubating at 99°C for 5 min, and slowly cooling down to 20°C within 15 min in a thermocycler.
T386 53112-53324 Sentence denotes Transposons were assembled by mixing Tn5(E54K, L372P) transposase (100 ng/μl) [purified according to (41)] with 1.25 μM annealed adapters in 50 mM Tris-HCl (pH 7.5) and incubating the reaction for 1 hour at 23°C.
T387 53325-53669 Sentence denotes Tagmentation was carried out by mixing 1.2 μl of the RT-LAMP product (~200 ng DNA) with 1.5 μl of loaded transposase in freshly prepared tagmentation buffer [10 mM [tris(hydroxymethyl)methylamino]propanesulfonic acid) (TAPS)] (pH 8.5), 5 mM MgCl2, and 10% (v/v) dimethylformamide] using a Liquidator 96 Manual Pipetting System (Mettler Toledo).
T388 53670-53718 Sentence denotes The reactions were incubated at 55°C for 10 min.
T389 53719-53791 Sentence denotes Reactions were stopped by adding SDS to a final concentration of 0.033%.
T390 53792-53958 Sentence denotes Tagmented DNA of each plate was pooled and size-selected using a two-step AMPureXP bead (Beckman Coulter) purification to target for fragments between 300 and 600 bp.
T391 53959-54075 Sentence denotes First, 50 μl of pooled reaction was mixed with 50 μl of water and bound to 55 μl of beads to remove large fragments.
T392 54076-54305 Sentence denotes To further remove small fragments, the supernatant of this reaction was added to 25 μl of fresh beads and further purified using two washes with 80% ethanol before the samples were finally eluted in 10 μl of 5 mM tris-HCl (pH 8).
T393 54306-54700 Sentence denotes One PCR per plate with 1 μl of the eluate and RT-LAMP–specific and Tn5-adapter–specific primers (P7nxt-GeneN-A-LBrc and P7-xi7001..7016, P5.fw) was performed using NEBNext Q5 HotStart polymerase (New England Biolabs) with two cycles at 62°C for annealing and 90 s elongation, followed by two cycles at 65°C for annealing and 90 s elongation, and 13 cycles at 72°C annealing and 90 s elongation.
T394 54701-54872 Sentence denotes All PCR reactions were combined and 19% of this pool was size-selected for 400 to 550 bp using a 2% agarose/tris-acetate-EDTA gel and column purification (Macherey-Nagel).
T395 54873-55213 Sentence denotes The final sequencing library was quantified by qPCR (New England Biolabs) and sequenced with a paired-end sequencing run on a NextSeq 550 machine (Illumina) with 20% phiX spike-in and 136 cycles for the first read, 11 cycles to read the 11-nt-long plate index (i7) and 20 cycles to read the 11-nt-long well index (i5) and the 9-nt-long UMI.
T396 55214-55327 Sentence denotes For trimming of the reads (i.e., removal of P7 Illumina adapter sequences), cutadapt (version 2.8) (42) was used.
T397 55328-55464 Sentence denotes For validation of the origin of the sequence of the LAMP product (fig. S4A), 107 reads were randomly selected and used for the analysis.
T398 55465-55602 Sentence denotes Reads were mapped to the SARS-CoV-2 reference genome (NC_045512.2) (43), using bwa-mem with default settings (version 0.7.17-r1188) (44).
T399 55603-55692 Sentence denotes Virus genome coverage was determined with the samtools depth command (version 1.10) (45).
T400 55693-55778 Sentence denotes Using bwa-mem, 80.6% of reads could be mapped to the virus genome (fig. S4, B and C).
T401 55779-55868 Sentence denotes To analyze the remaining sequences, a k-mer analysis using a custom script was performed.
T402 55869-56044 Sentence denotes Using 9-mers, this matched 93.5% of the nonmapped reads with a maximal Levenshtein distance of two to one of the LAMP primers or their reverse complement sequences (fig. S4D).
T403 56045-56141 Sentence denotes This is explained by the fact that LAMP products can consist of complex sequence rearrangements.
T404 56142-56254 Sentence denotes For classification of samples by LAMP-sequencing, reads were assigned to wells and counted using custom scripts.
T405 56255-56478 Sentence denotes A read was considered as a match to SARS-CoV-2 N gene if at least one of three short sequences (~13 nt, marked orange in fig. S4A) not covered by RT-LAMP primers was found in the read, otherwise it was counted as unmatched.
T406 56479-56596 Sentence denotes Sequencing reads were grouped by UMI and by position of the matched sequence with the aim of removing PCR duplicates.
T407 56597-56737 Sentence denotes A sample was considered if more than 200 total UMIs were observed and called positive if more than 10,000 virus-matching UMIs were observed.
T408 56738-56850 Sentence denotes There is a very wide gap in the number of virus-matching reads between positive and negative samples (fig. S5A):
T409 56851-56908 Sentence denotes The count is either below 7000 UMIs or above 45,000 UMIs.
T410 56909-57019 Sentence denotes This is why we placed the decision threshold for scoring a sample as LAMP-sequencing positive within this gap.
T411 57020-57309 Sentence denotes The fact that also RT-qPCR–negative samples give rise to some UMI counts containing viral sequences is explained by template switching of unattached adapters that remain in the reaction after tagmentation, but no cause for concern due to the wide gap between negative and positive samples.
T412 57310-57456 Sentence denotes For a few samples, we saw so few reads (less than 200 UMIs) that we suspected that the multiplexing had failed and excluded them from the results.
T413 57457-57635 Sentence denotes As most of these were in the same row of the same plate, we analyzed these samples after LAMP-sequencing by gel electrophoresis (fig. S5B) to check for DNA content after RT-LAMP.
T414 57636-57780 Sentence denotes We found that the gel results agree with the RT-LAMP outcome, indicating that the failure likely was caused later, probably during multiplexing.
T415 57782-57802 Sentence denotes Statistical analysis
T416 57803-57940 Sentence denotes Except where otherwise noted, all data were analyzed with R (46) using the tidyverse (47) and ggplot2 (48) system or with GraphPad Prism.
T417 57941-58019 Sentence denotes Sensitivity and specificity values were obtained from count tables as follows:
T418 58020-58157 Sentence denotes Specificity of the RT-LAMP assay was calculated as the fraction of RT-qPCR–negative samples that were also negative in the RT-LAMP assay.
T419 58158-58321 Sentence denotes Sensitivity for a given CT interval was calculated as the fraction of all samples with an RT-qPCR CT value in that interval that was positive in the RT-LAMP assay.
T420 58322-58544 Sentence denotes In both cases, 95% confidence intervals were calculated by interpreting the fractions of counts as binomial rates and then using Wilson’s method for binomial confidence intervals as implemented in the R package binom (49).
T421 58545-58709 Sentence denotes The R code used to perform analyses and produce figures can be found on GitHub, together with all data tables: https://github.com/anders-biostat/LAMP-Paper-Figures.
T422 58711-58726 Sentence denotes Acknowledgments
T423 58727-58738 Sentence denotes We thank V.
T424 58739-58752 Sentence denotes Pelechano, X.
T425 58753-58764 Sentence denotes Yin, and V.
T426 58765-58826 Sentence denotes Lohmann for helpful discussions and sharing unpublished work.
T427 58827-58838 Sentence denotes We thank U.
T428 58839-59045 Sentence denotes Merle for providing specimens and the diagnostic laboratory team at Heidelberg University Hospital for providing RNA samples isolated from pharyngeal swabs collected from patients being tested for COVID-19.
T429 59046-59057 Sentence denotes We thank V.
T430 59058-59077 Sentence denotes Sonntag-Buck, A.-M.
T431 59078-59088 Sentence denotes Heuser, I.
T432 59089-59099 Sentence denotes Ambiel, S.
T433 59100-59110 Sentence denotes Winter, T.
T434 59111-59125 Sentence denotes Müller, and S.
T435 59126-59165 Sentence denotes Horner for excellent technical support.
T436 59166-59177 Sentence denotes We thank A.
T437 59178-59195 Sentence denotes Uvarovskii and S.
T438 59196-59235 Sentence denotes Ovchinnikova for IT-related help and S.
T439 59236-59249 Sentence denotes Kreger and U.
T440 59250-59286 Sentence denotes Jäkle for additional technical help.
T441 59287-59379 Sentence denotes Funding: V.L.D.T., A.F., and P.C. were supported by the Chica and Heinz Schaller foundation.
T442 59380-59453 Sentence denotes M.K. was supported by the Deutsche Forschungsgemeinschaft (DFG; grant no.
T443 59454-59466 Sentence denotes KN498/11-1).
T444 59467-59523 Sentence denotes S.B. was supported by the Heisenberg program of the DFG.
T445 59524-59612 Sentence denotes Author contributions: V.L.D.T., H.-G.K., M.K., and S.A. conceived and planned the study.
T446 59613-59773 Sentence denotes V.L.D.T., M.M., K.B., and M.K. planned and designed the experiments on the RT-LAMP assay and performed them together with Daniel Kirrmaier, A.F., S.K., and P.C.
T447 59774-59855 Sentence denotes V.L.D.T., K.B., and M.K. performed the experiments for the swab–to–RT-LAMP assay.
T448 59856-59943 Sentence denotes K.H. designed the LAMP-sequencing assay and performed it with Daniel Kirrmaier and M.K.
T449 59944-60114 Sentence denotes K.B. performed the RT-qPCR analyses of samples and organized, together with I.B.M., P.S., Dina Khalid, and H.-G.K., provision of the samples by the diagnostic laboratory.
T450 60115-60177 Sentence denotes D.P. and C.G. tested sample handling and logistics strategies.
T451 60178-60247 Sentence denotes V.L.D.T., M.K., M.L.S., and S.B. tested viral inactivation protocols.
T452 60248-60336 Sentence denotes S.A. performed data analysis of the RT-LAMP experiments, together with L.P.M.K. and K.H.
T453 60337-60376 Sentence denotes K.H. analyzed the LAMP-sequencing data.
T454 60377-60420 Sentence denotes V.L.D.T. analyzed the swab–to–RT-LAMP data.
T455 60421-60502 Sentence denotes V.L.D.T., K.H., P.C., L.P.M.K., M.K., and S.A. analyzed and interpreted the data.
T456 60503-60584 Sentence denotes V.L.D.T., K.H., M.K., and S.A. wrote the manuscript, with input from all authors.
T457 60585-60637 Sentence denotes Competing interests: P.S. has consulted for Janssen.
T458 60638-60702 Sentence denotes The other authors declare that they have no competing interests.
T459 60703-60735 Sentence denotes Data and materials availability:
T460 60736-60824 Sentence denotes All data associated with this study are in the main text or the Supplementary Materials.
T461 60825-60993 Sentence denotes The R code used to perform analyses and produce figures can be found on GitHub, together with all raw data tables: https://github.com/anders-biostat/LAMP-Paper-Figures.
T462 60994-61215 Sentence denotes This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
T463 61216-61298 Sentence denotes To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
T464 61299-61500 Sentence denotes This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using this material.
T465 61502-61525 Sentence denotes SUPPLEMENTARY MATERIALS
T466 61526-61586 Sentence denotes stm.sciencemag.org/cgi/content/full/scitranslmed.abc7075/DC1
T467 61587-61608 Sentence denotes Materials and Methods
T468 61609-61617 Sentence denotes Fig. S1.
T469 61618-61638 Sentence denotes Design of the study.
T470 61639-61647 Sentence denotes Fig. S2.
T471 61648-61750 Sentence denotes Comparison of the RT-LAMP assay with CT values from RT-qPCR using primer sets E-Sarbeco and N-Sarbeco.
T472 61751-61759 Sentence denotes Fig. S3.
T473 61760-61869 Sentence denotes Comparison of the RT-LAMP assay using primer set 1a-A with CT values from RT-qPCR using primer set E-Sarbeco.
T474 61870-61878 Sentence denotes Fig. S4.
T475 61879-61913 Sentence denotes Analysis of LAMP-sequencing reads.
T476 61914-61922 Sentence denotes Fig. S5.
T477 61923-61966 Sentence denotes Sample classification with LAMP-sequencing.
T478 61967-61975 Sentence denotes Fig. S6.
T479 61976-62073 Sentence denotes RNA stability and detection limit of the RT-LAMP assay in pharyngeal swab specimens with IVT RNA.
T480 62074-62082 Sentence denotes Fig. S7.
T481 62083-62146 Sentence denotes Swab–to–RT-LAMP assay titration of positive COVID-19 specimens.
T482 62147-62156 Sentence denotes Table S1.
T483 62157-62211 Sentence denotes Sequences of primers and amplicons used in this study.
T484 62212-62221 Sentence denotes Table S2.
T485 62222-62283 Sentence denotes Sensitivity and specificity of the RT-LAMP test from Fig. 3B.
T486 62284-62293 Sentence denotes Table S3.
T487 62294-62355 Sentence denotes Sensitivity and specificity of the RT-LAMP test from Fig. 5B.
T488 62356-62365 Sentence denotes Table S4.
T489 62366-62399 Sentence denotes Primers used for LAMP-sequencing.
T490 62400-62409 Sentence denotes Table S5.
T491 62410-62474 Sentence denotes Overview of time requirements for various sample handling steps.
T492 62475-62488 Sentence denotes Data File S1.
T493 62489-62507 Sentence denotes Raw data for figs.
T494 62508-62518 Sentence denotes S6 and S7.
T495 62520-62577 Sentence denotes View/request a protocol for this paper from Bio-protocol.