LitCovid-sentences  

PMC:7574920 / 2878-58709 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T21 0-12 Sentence denotes INTRODUCTION
T22 13-187 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 188-399 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 400-642 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 643-767 Sentence denotes This suggests that infection control strategies focusing on symptomatic patients are not sufficient to prevent virus spread.
T26 768-915 Sentence denotes Therefore, large-scale diagnostic methods are needed to determine the spread of the virus in populations quickly, comprehensively, and sensitively.
T27 916-1014 Sentence denotes This would allow for the rapid isolation of infected persons during an existing wave of infection.
T28 1015-1252 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 1253-1385 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 1386-1587 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 1588-1673 Sentence denotes Therefore, an assay that detects SARS-CoV-2 RNA facilitates testing of large cohorts.
T32 1674-1952 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 1953-2169 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 2170-2336 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 2337-2588 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 2589-2782 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 2783-2907 Sentence denotes Therefore, increasing daily test capacities for RT-qPCR–based diagnostics for SARS-CoV-2 RNA detection is currently limited.
T38 2908-3038 Sentence denotes To accelerate and optimize such diagnostics, new scalable methods for RNA isolation and the detection of viral genomes are needed.
T39 3039-3145 Sentence denotes An alternative to RT-qPCR is reverse transcription loop-mediated isothermal amplification (RT-LAMP) (5–7).
T40 3146-3359 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 3360-3498 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 3499-3678 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 3679-3750 Sentence denotes These, in turn, serve as self-priming templates for the DNA polymerase.
T44 3751-3939 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 3940-4023 Sentence denotes To detect DNA production in RT-LAMP assays, various approaches have been described.
T46 4024-4138 Sentence denotes One possibility is to use a pH indicator (e.g., phenol red) and run the reaction in a weakly buffered environment.
T47 4139-4310 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 4311-4432 Sentence denotes Previously, RT-LAMP assays have been proposed for diagnostic detection of other RNA viruses, such as influenza virus (9).
T49 4433-4555 Sentence denotes Also, several studies have demonstrated the use of isothermal DNA amplification to detect small amounts of SARS-CoV-2 RNA.
T50 4556-4755 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 4756-5008 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 5009-5149 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 5150-5430 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 5431-5576 Sentence denotes For detection, simple mobile phone cameras, copy machines, office scanners, or plate scanners with spectrophotometric quantification can be used.
T55 5577-6029 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 6030-6168 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 6169-6331 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 6332-6568 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 6570-6577 Sentence denotes RESULTS
T60 6579-6674 Sentence denotes Establishing colorimetric RT-LAMP assay sensitivity using an artificial SARS-CoV-2 RNA template
T61 6675-6810 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 6811-6929 Sentence denotes This mix contains two enzymes, an engineered reverse transcriptase (RTx) and a strand-displacing polymerase (Bst 2.0).
T63 6930-7181 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 7182-7394 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 7395-7591 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 7592-7792 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 7793-7845 Sentence denotes The reaction was conducted for up to 1 hour at 65°C.
T68 7846-7937 Sentence denotes For time points > 30 to 35 min, the negative control frequently became yellowish (Fig. 1A).
T69 7938-8038 Sentence denotes This was caused by spurious amplification products, which is a well-known problem with RT-LAMP (14).
T70 8039-8243 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 8244-8310 Sentence denotes Fig. 1 Sensitivity of the RT-LAMP assay determined using IVT RNA.
T72 8311-8457 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 8458-8561 Sentence denotes At indicated times, samples were removed from the heating block and cooled on ice to stop the reaction.
T74 8562-8780 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 8781-8991 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 8993-9057 Sentence denotes Testing clinical RNA samples with the colorimetric RT-LAMP assay
T77 9058-9185 Sentence denotes To evaluate the colorimetric RT-LAMP assay, we needed to compare its sensitivity and specificity to a validated RT-qPCR method.
T78 9186-9310 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 9311-9442 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 9443-9719 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 9720-9971 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 9972-10232 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 10233-10333 Sentence denotes Fig. 2 Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.
T84 10334-10446 Sentence denotes RNA samples isolated from 95 pharyngeal swab specimens were analyzed by the RT-LAMP assay using a 96-well plate.
T85 10447-10676 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 10677-10919 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 10920-11087 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 11088-11146 Sentence denotes Yellow (positive) samples yield a ΔOD of about 0.3 to 0.4.
T89 11147-11179 Sentence denotes Each line represents one sample.
T90 11180-11416 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 11417-11447 Sentence denotes Each dot is one sample (well).
T92 11448-11596 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 11597-11774 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 11775-11893 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 11894-12185 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 12186-12292 Sentence denotes This suggested a detection limit of the colorimetric RT-LAMP assay corresponding to a CT ≈ 30 for RT-qPCR.
T97 12293-12488 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 12489-12572 Sentence denotes The performance of each RT-qPCR run was validated using this as a positive control.
T99 12573-12813 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 12814-12990 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 12991-13285 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 13286-13433 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 13434-13582 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 13583-13706 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 13707-13845 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 13846-14113 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 14114-14284 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 14286-14359 Sentence denotes Validation of the colorimetric RT-LAMP assay for SARS-CoV-2 RNA detection
T109 14360-14468 Sentence denotes To determine the specificity and sensitivity of the RT-LAMP assay, we continued to analyze more RNA samples.
T110 14469-14544 Sentence denotes We assayed a total of 768 RNA samples obtained on different days (fig. S1).
T111 14545-14831 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 14832-14892 Sentence denotes Fig. 3 Detection of SARS-CoV-2 RNA using the RT-LAMP assay.
T113 14893-15018 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 15019-15133 Sentence denotes The RNA extraction method (QC, QiaCube, a column-based method; QS, QiaSymphony, a bead-based method) is indicated.
T115 15134-15249 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 15250-15305 Sentence denotes The 96-well plate shown in Fig. 2 is not included here.
T117 15306-15531 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 15532-15646 Sentence denotes The specificity is the fraction of RT-qPCR–negative samples correctly identified as negative by the RT-LAMP assay.
T119 15647-15940 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 15941-16182 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 16183-16297 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 16298-16389 Sentence denotes Fig. 3B and table S2 show specificity and sensitivity values calculated from these numbers.
T123 16390-16397 Sentence denotes RT-LAMP
T124 16398-16417 Sentence denotes CT Pos Neg Sum
T125 16418-16449 Sentence denotes RT-qPCR Pos 0–25 51 0 51
T126 16450-16466 Sentence denotes 25–30 28 2 30
T127 16467-16483 Sentence denotes 30–35 4 16 20
T128 16484-16500 Sentence denotes 35–40 0 16 16
T129 16501-16523 Sentence denotes Neg Neg 2 649 651
T130 16524-16717 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 16718-16917 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 16918-17040 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 17041-17199 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 17200-17363 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 17364-17452 Sentence denotes The overall specificity of the RT-LAMP test was 99.7% (Wilson’s 95% confidence interval:
T136 17453-17569 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 17570-17608 Sentence denotes 91.4 to 99.3%) (Fig. 3B and table S2).
T138 17610-17661 Sentence denotes Multiplexed sequencing of RT-LAMP reaction products
T139 17662-17811 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 17812-17945 Sentence denotes Validation of positive results, however, required confirmation that the RT-LAMP reaction led to the amplification of viral sequences.
T141 17946-18082 Sentence denotes To analyze the sequences of many RT-LAMP reaction products, we established multiplexed sequencing of RT-LAMP products (LAMP-sequencing).
T142 18083-18166 Sentence denotes LAMP-sequencing is based on Tn5 transposase tagmentation (17) and sample barcoding.
T143 18167-18288 Sentence denotes Tagmentation enables fragmentation and direct adapter ligation of DNA samples for analysis by next-generation sequencing.
T144 18289-18407 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 18408-18544 Sentence denotes After tagmentation, all barcoded fragments from each plate were pooled and size-selected by bead purification to remove excess adapters.
T146 18545-18656 Sentence denotes A second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented RT-LAMP fragments.
T147 18657-18833 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 18834-18912 Sentence denotes Fig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).
T149 18913-18955 Sentence denotes (A) Workflow for LAMP-sequencing is shown.
T150 18956-19134 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 19135-19251 Sentence denotes After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection.
T152 19252-19597 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 19598-19629 Sentence denotes Each dot represents one sample.
T154 19630-19789 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 19790-20092 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 20093-20274 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 20275-20478 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 20479-20548 Sentence denotes This indicated that LAMP-sequencing amplified the targeted sequences.
T159 20549-20715 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 20716-21014 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 21015-21093 Sentence denotes For 754 of the 768 samples, we obtained enough reads to make a call (fig. S5).
T162 21094-21270 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 21271-21468 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 21469-21513 Sentence denotes Table 2 Summary of LAMP-sequencing results.
T165 21514-21768 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 21769-21776 Sentence denotes RT-LAMP
T167 21777-21796 Sentence denotes CT Pos Neg Sum
T168 21797-21854 Sentence denotes LAMP- sequencing Pos RT-qPCR Pos 0–25 49 0 49
T169 21855-21871 Sentence denotes 25–30 28 0 28
T170 21872-21886 Sentence denotes 30–35 4 0 4
T171 21887-21901 Sentence denotes 35–40 0 0 0
T172 21902-21920 Sentence denotes Neg Neg 0 0 0
T173 21921-21956 Sentence denotes Neg RT-qPCR Pos 0–25 0 0 0
T174 21957-21971 Sentence denotes 25–30 0 2 2
T175 21972-21988 Sentence denotes 30–35 0 16 16
T176 21989-22005 Sentence denotes 35–40 0 16 16
T177 22006-22028 Sentence denotes Neg Neg 2 637 639
T178 22029-22074 Sentence denotes Too few reads RT-qPCR Pos 0–25 2 0 2
T179 22075-22089 Sentence denotes 25–30 0 0 0
T180 22090-22104 Sentence denotes 30–35 0 0 0
T181 22105-22119 Sentence denotes 35–40 0 0 0
T182 22120-22140 Sentence denotes Neg Neg 0 12 12
T183 22141-22262 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 22263-22375 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 22376-22434 Sentence denotes LAMP-sequencing eliminated all of these samples (Fig. 4C).
T186 22435-22674 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 22675-22876 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 22878-22923 Sentence denotes A swab–to–RT-LAMP assay without RNA isolation
T189 22924-23039 Sentence denotes RNA isolation is time consuming, costly, and depends on reagents with potentially limited supply during a pandemic.
T190 23040-23253 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 23254-23472 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 23473-23665 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 23666-23798 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 23799-23995 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 23996-24025 Sentence denotes S6 and S7, and data file S1).
T196 24026-24354 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 24356-24411 Sentence denotes Testing clinical samples with the swab–to–RT-LAMP assay
T198 24412-24629 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 24630-24741 Sentence denotes As an additional precaution, we kept the samples in the cold (using an ice-cold metal block) whenever possible.
T200 24742-24849 Sentence denotes For testing large numbers of clinical samples, we performed the RT-LAMP assay using several 96-well plates.
T201 24850-25008 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 25009-25156 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 25157-25277 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 25278-25520 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 25521-25665 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 25666-25770 Sentence denotes We found that some positive samples did not induce a color change but did so when assayed a second time.
T207 25771-25846 Sentence denotes We therefore would recommend running this assay using technical duplicates.
T208 25847-25913 Sentence denotes Fig. 5 Swab–to–RT-LAMP assay of clinical pharyngeal swab samples.
T209 25914-26087 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 26088-26226 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 26227-26499 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 26500-26741 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 26742-26856 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 26857-26948 Sentence denotes Fig. 5A and table S3 show specificity and sensitivity values calculated from these numbers.
T215 26949-26979 Sentence denotes Hot swab–to–RT-LAMP RT-LAMP
T216 26980-27000 Sentence denotes CT Pos Neg Sum
T217 27001-27032 Sentence denotes RT-qPCR Pos 0–25 38 4 42
T218 27033-27049 Sentence denotes 25–30 17 5 22
T219 27050-27066 Sentence denotes 30–35 5 23 28
T220 27067-27083 Sentence denotes 35–40 0 36 36
T221 27084-27106 Sentence denotes Neg Neg 1 214 215
T222 27107-27124 Sentence denotes Sum 61 282 343
T223 27125-27158 Sentence denotes Direct swab–to–RT-LAMP RT-LAMP
T224 27159-27179 Sentence denotes CT Pos Neg Sum
T225 27180-27211 Sentence denotes RT-qPCR Pos 0–25 15 1 16
T226 27212-27228 Sentence denotes 25–30 6 11 17
T227 27229-27245 Sentence denotes 30–35 2 21 23
T228 27246-27262 Sentence denotes 35–40 3 23 26
T229 27263-27285 Sentence denotes Neg Neg 9 144 153
T230 27286-27303 Sentence denotes Sum 35 200 235
T231 27305-27362 Sentence denotes Heterogeneity of specimen pH in the swab–to–RT-LAMP assay
T232 27363-27573 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 27574-27680 Sentence denotes This was likely due to a sample-specific variability that influenced the starting pH in the LAMP reaction.
T234 27681-27767 Sentence denotes This might have affected the interpretability of the measurement at 30 min (ΔOD30min).
T235 27768-27831 Sentence denotes We investigated how this pH shift influenced the RT-LAMP assay.
T236 27832-27964 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 27965-28147 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 28148-28330 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 28331-28400 Sentence denotes Fig. 6 Colorimetric readouts of the swab–to–RT-LAMP assay over time.
T240 28401-28525 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 28526-28663 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 28664-28724 Sentence denotes Also, the 40-min time point was not available for one plate.
T243 28725-28850 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 28851-29152 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 29153-29226 Sentence denotes The latter shows better separation between positive and negative samples.
T246 29227-29372 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 29373-29538 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 29539-29711 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 29713-29723 Sentence denotes DISCUSSION
T250 29724-29830 Sentence denotes Here, we evaluated the use and suitability of the RT-LAMP assay for the detection of SARS-CoV-2 infection.
T251 29831-29969 Sentence denotes We also developed LAMP-sequencing as a fully scalable alternative to colorimetric or fluorometric analysis of DNA amplification reactions.
T252 29970-30181 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 30182-30345 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 30346-30614 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 30615-30683 Sentence denotes For samples with CT > 30, the RT-LAMP assay was much less sensitive.
T256 30684-30801 Sentence denotes However, there is debate about which CT value for a positive RT-qPCR result should be considered clinically relevant.
T257 30802-30900 Sentence denotes Vogels et al. (16) indicate that a CT value above 36 corresponds to less than 10 molecules of RNA.
T258 30901-31067 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 31068-31327 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 31328-31589 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 31590-31680 Sentence denotes Furthermore, alternative sample types, e.g., sputum or stool (29), might be more reliable.
T262 31681-31907 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 31908-32023 Sentence denotes Compatibility of the RT-LAMP assay with direct saliva specimens has been shown using spike-in experiments (22, 34).
T264 32024-32171 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 32172-32434 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 32435-32652 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 32653-32908 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 32909-33054 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 33055-33252 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 33253-33432 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 33433-33637 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 33638-33667 Sentence denotes S6 and S7, and data file S1).
T273 33668-33843 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 33844-34028 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 34029-34126 Sentence denotes However, these applications have yet to be tested with large numbers of diverse clinical samples.
T276 34127-34195 Sentence denotes There are several differences between the RT-LAMP assay and RT-qPCR.
T277 34196-34425 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 34426-34500 Sentence denotes This makes the RT-LAMP assay more amenable for point-of-care applications.
T279 34501-34618 Sentence denotes Second, the reagents for the RT-LAMP assay are different from the ones used for RT-qPCR and are supplier independent.
T280 34619-34789 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 34790-34964 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 34965-35102 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 35103-35206 Sentence denotes The RT-LAMP assay overcomes this problem and therefore will be a more scalable method for mass testing.
T284 35208-35273 Sentence denotes Application of RT-LAMP and LAMP-sequencing for SARS-CoV-2 testing
T285 35274-35381 Sentence denotes With its good sensitivity for samples up to CT ≈ 30, the colorimetric RT-LAMP assay has several advantages:
T286 35382-35453 Sentence denotes It is fast, inexpensive, and it can be evaluated without any equipment.
T287 35454-35702 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 35703-35867 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 35868-36145 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 36146-36219 Sentence denotes It would be desirable to have a similar precaution for the RT-LAMP assay.
T291 36220-36348 Sentence denotes A multiplexed fluorescence readout might provide this (34) but comes at the expense of the simplicity of a colorimetric readout.
T292 36349-36643 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 36644-36830 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 36831-36975 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 36976-37211 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 37212-37319 Sentence denotes Thus, LAMP-sequencing could become an important part of workflows for routine testing of large populations.
T297 37320-37531 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 37532-37757 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 37758-37801 Sentence denotes There are several limitations to our study.
T300 37802-37912 Sentence denotes We used surplus RNA sample material from a diagnostic laboratory rather than newly collected clinical samples.
T301 37913-38016 Sentence denotes The criteria for testing individuals may have influenced cohort characteristics and hence our findings.
T302 38017-38188 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 38189-38400 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 38401-38601 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 38602-38801 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 38802-38904 Sentence denotes We used LAMP-sequencing to validate the RT-LAMP assay results and did not use it as a diagnostic tool.
T307 38905-39082 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 39083-39204 Sentence denotes Also, reagents such as the primer sets for the RT-LAMP assay may be subject to production-dependent quality fluctuations.
T309 39205-39318 Sentence denotes Therefore, all reagents must be precisely validated (batch control) before using an RT-LAMP assay diagnostically.
T310 39319-39433 Sentence denotes Application of the RT-LAMP assay has great potential, even more so as more sensitive primer sets become available.
T311 39434-39574 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 39575-39773 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 39774-39998 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 40000-40021 Sentence denotes MATERIALS AND METHODS
T315 40023-40035 Sentence denotes Study design
T316 40036-40221 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 40222-40433 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 40434-40640 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 40641-40815 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 40816-41069 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 41070-41145 Sentence denotes This study was conducted in Heidelberg, Germany in March and April of 2020.
T322 41146-41424 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 41425-41699 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 41700-41849 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 41850-42029 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 42031-42055 Sentence denotes Clinical sample handling
T327 42056-42169 Sentence denotes Specimens were collected as nasopharyngeal and oropharyngeal flocked swabs in Amies medium (eSwab, Copan Italia).
T328 42170-42342 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 42343-42529 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 42530-42641 Sentence denotes Samples were processed in a biosafety level 2 cabinet until inactivation by heat or mixing with a lysis buffer.
T331 42643-42668 Sentence denotes RNA isolation and RT-qPCR
T332 42669-43016 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 43017-43197 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 43198-43345 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 43346-43502 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 43503-43716 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 43717-43833 Sentence denotes 53-0776-96; TIB MOLBIOL Syntheselabor GmbH, Berlin, Germany) or LightMix Modular SARS and Wuhan CoV N gene (cat. no.
T338 43834-43923 Sentence denotes 53-0775-96; TIB MOLBIOL), 0.5 μl of LightMix Modular EAV RNA Extraction Control (cat. no.
T339 43924-44061 Sentence denotes 66-0909-96; TIB MOLBIOL), and 0.1 μl of reverse transcriptase enzyme (LightCycler Multiplex RNA Virus Master, Roche, Basel, Switzerland).
T340 44062-44180 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 44181-44266 Sentence denotes The performance of the RT-qPCR was validated using a positive control for the E gene.
T342 44267-44351 Sentence denotes A total of 103 molecules of E gene RNA per RT-qPCR reaction correspond to a CT ≈ 30.
T343 44353-44395 Sentence denotes RT-LAMP primer design and positive control
T344 44396-44611 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 44612-44747 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 44748-45121 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 45123-45206 Sentence denotes Liquid handling using 96-well plates and precautions taken to prevent contamination
T348 45207-45273 Sentence denotes To prevent cross-contamination, we have taken several precautions.
T349 45274-45380 Sentence denotes The 10× primer mix was prepared with nuclease-free water (AM9937, Ambion) and stored in aliquots at −20°C.
T350 45381-45549 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 45550-45670 Sentence denotes The 96-well PCR plate containing the RT-LAMP mix was covered with an Society for Biomolecular Screening (SBS) plate lid.
T352 45671-45816 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 45817-45988 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 45989-46208 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 46209-46423 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 46425-46438 Sentence denotes RT-LAMP assay
T357 46439-46580 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 46581-46915 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 46916-46950 Sentence denotes Values given are for one reaction:
T360 46951-47230 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 47231-47412 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 47413-47596 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 47597-47747 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 47748-47798 Sentence denotes This intensifies the color before the measurement.
T365 47799-47899 Sentence denotes Photographs were taken with cell phone cameras or the scanner function of an office copying machine.
T366 47901-47939 Sentence denotes Quantification of the RT-LAMP reaction
T367 47940-48056 Sentence denotes Absorbance measurements were performed with a Spark Cyto or Infinite M200 (Tecan) at 434 and 560 nm with 25 flashes.
T368 48057-48217 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 48218-48324 Sentence denotes To obtain a good readout of the color change, absorbance at 560 nm was substracted from the one at 434 nm.
T370 48325-48357 Sentence denotes This difference was denoted ΔOD.
T371 48359-48380 Sentence denotes Swab–to–RT-LAMP assay
T372 48381-48496 Sentence denotes For direct and hot swab–to–RT-LAMP assays, patient swab specimens were transferred first onto a 96-well seed plate.
T373 48497-48657 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 48658-48776 Sentence denotes The plate was sealed using a transparent adhesive foil (GK480-OS, Kisker Biotech) and kept on an ice-cold metal block.
T375 48777-48958 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 48959-49024 Sentence denotes The seed plate was cooled down to 4°C on an ice-cold metal block.
T377 49025-49158 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 49159-49244 Sentence denotes This plate was also sealed with transparent adhesive foil (GK480-OS, Kisker Biotech).
T379 49245-49318 Sentence denotes Both plates were then incubated at 65°C for the LAMP reaction to proceed.
T380 49319-49480 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 49481-49631 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 49633-49655 Sentence denotes LAMP-sequencing method
T383 49656-49876 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 49877-49925 Sentence denotes The relevant primers are summarized in table S4.
T385 49926-50233 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 50234-50446 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 50447-50791 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 50792-50840 Sentence denotes The reactions were incubated at 55°C for 10 min.
T389 50841-50913 Sentence denotes Reactions were stopped by adding SDS to a final concentration of 0.033%.
T390 50914-51080 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 51081-51197 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 51198-51427 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 51428-51822 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 51823-51994 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 51995-52335 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 52336-52449 Sentence denotes For trimming of the reads (i.e., removal of P7 Illumina adapter sequences), cutadapt (version 2.8) (42) was used.
T397 52450-52586 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 52587-52724 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 52725-52814 Sentence denotes Virus genome coverage was determined with the samtools depth command (version 1.10) (45).
T400 52815-52900 Sentence denotes Using bwa-mem, 80.6% of reads could be mapped to the virus genome (fig. S4, B and C).
T401 52901-52990 Sentence denotes To analyze the remaining sequences, a k-mer analysis using a custom script was performed.
T402 52991-53166 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 53167-53263 Sentence denotes This is explained by the fact that LAMP products can consist of complex sequence rearrangements.
T404 53264-53376 Sentence denotes For classification of samples by LAMP-sequencing, reads were assigned to wells and counted using custom scripts.
T405 53377-53600 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 53601-53718 Sentence denotes Sequencing reads were grouped by UMI and by position of the matched sequence with the aim of removing PCR duplicates.
T407 53719-53859 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 53860-53972 Sentence denotes There is a very wide gap in the number of virus-matching reads between positive and negative samples (fig. S5A):
T409 53973-54030 Sentence denotes The count is either below 7000 UMIs or above 45,000 UMIs.
T410 54031-54141 Sentence denotes This is why we placed the decision threshold for scoring a sample as LAMP-sequencing positive within this gap.
T411 54142-54431 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 54432-54578 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 54579-54757 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 54758-54902 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 54904-54924 Sentence denotes Statistical analysis
T416 54925-55062 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 55063-55141 Sentence denotes Sensitivity and specificity values were obtained from count tables as follows:
T418 55142-55279 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 55280-55443 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 55444-55666 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 55667-55831 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.