PMC:7574920 / 9448-32589 JSON TXT

Annnotations TAB JSON ListView MergeView

LitCovid-PD-FMA-UBERON

LitCovid-PD-MONDO

LitCovid-PD-CLO

LitCovid-PD-CHEBI

LitCovid-PubTator

LitCovid-PD-GO-BP

LitCovid-PD-GlycoEpitope

{"project":"LitCovid-PD-GlycoEpitope","denotations":[{"id":"T2","span":{"begin":11541,"end":11544},"obj":"GlycoEpitope"},{"id":"T3","span":{"begin":12514,"end":12517},"obj":"GlycoEpitope"}],"attributes":[{"id":"A2","pred":"glyco_epitope_db_id","subj":"T2","obj":"http://www.glycoepitope.jp/epitopes/AN0083"},{"id":"A3","pred":"glyco_epitope_db_id","subj":"T3","obj":"http://www.glycoepitope.jp/epitopes/AN0083"}],"text":"RESULTS\n\nEstablishing colorimetric RT-LAMP assay sensitivity using an artificial SARS-CoV-2 RNA template\nTo 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. This mix contains two enzymes, an engineered reverse transcriptase (RTx) and a strand-displacing polymerase (Bst 2.0). 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. 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. 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). 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. The reaction was conducted for up to 1 hour at 65°C. For time points \u003e 30 to 35 min, the negative control frequently became yellowish (Fig. 1A). This was caused by spurious amplification products, which is a well-known problem with RT-LAMP (14). 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).\nFig. 1 Sensitivity of the RT-LAMP assay determined using IVT RNA.\n(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. At indicated times, samples were removed from the heating block and cooled on ice to stop the reaction. 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. 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.\n\nTesting clinical RNA samples with the colorimetric RT-LAMP assay\nTo evaluate the colorimetric RT-LAMP assay, we needed to compare its sensitivity and specificity to a validated RT-qPCR method. 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. 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). 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. 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). 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).\nFig. 2 Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.\nRNA samples isolated from 95 pharyngeal swab specimens were analyzed by the RT-LAMP assay using a 96-well plate. 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. 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. 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. Yellow (positive) samples yield a ΔOD of about 0.3 to 0.4. Each line represents one sample. 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. Each dot is one sample (well). In a colorimetric RT-LAMP reaction, positive samples with a CT \u003c 30 changed the color of the phenol-red dye within the first 30 min of the reaction. Samples with a CT \u003e 30 either did not change their color or did so at time points \u003e 35 min, simultaneously with a color change observed in some of the negative samples (Fig. 1). On the basis of this observation, we used the ΔOD value at 30 min to decide whether a sample was positive or negative. Plotting the ΔOD measurements versus CT values at the 30-min time point revealed that all patient samples with a CT \u003c 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). This suggested a detection limit of the colorimetric RT-LAMP assay corresponding to a CT ≈ 30 for RT-qPCR.\nThe 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). The performance of each RT-qPCR run was validated using this as a positive control. 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). 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.\nIn 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). 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). 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). This suggested that the N gene primers were less sensitive than the E gene primers for detecting SARS-CoV-2 RNA by RT-qPCR. Similar differences have been observed previously for other primer sets, e.g., between the E gene primers and the RdRp-SARSr primers (16).\nFor 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). 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.\n\nValidation of the colorimetric RT-LAMP assay for SARS-CoV-2 RNA detection\nTo determine the specificity and sensitivity of the RT-LAMP assay, we continued to analyze more RNA samples. We assayed a total of 768 RNA samples obtained on different days (fig. S1). 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.\nFig. 3 Detection of SARS-CoV-2 RNA using the RT-LAMP assay.\n(A) Scatter plot shows a comparison of RT-LAMP assay results and RT-qPCR results for RNA samples tested on 10 96-well plates. The RNA extraction method (QC, QiaCube, a column-based method; QS, QiaSymphony, a bead-based method) is indicated. The time point for measurement by the colorimetric RT-LAMP assay was 30 min after the start of the 65°C incubation. The 96-well plate shown in Fig. 2 is not included here. 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. The specificity is the fraction of RT-qPCR–negative samples correctly identified as negative by the RT-LAMP assay. 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. 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).\nTable 1 Shown is RT-qPCR and RT-LAMP testing of 768 clinical samples stratified into CT value bins (see Fig. 3A).\nFig. 3B and table S2 show specificity and sensitivity values calculated from these numbers.\nRT-LAMP\nCT Pos Neg Sum\nRT-qPCR Pos 0–25 51 0 51\n25–30 28 2 30\n30–35 4 16 20\n35–40 0 16 16\nNeg Neg 2 649 651\nSum 85 683 768 The consistency of the results during the analysis confirmed a threshold of ΔOD \u003e +0.3 as a robust measure to identify samples that were positive for SARS-CoV-2 RNA (Fig. 3A). RT-qPCR–positive samples with a CT \u003c 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). This confirmed the sensitivity of the RT-LAMP assay for detection of SARS-CoV-2 RNA in samples corresponding to a CT \u003c 30. We observed small differences between different plates on the exact sensitivity threshold, probably caused by slight variability in plate or reagent handling. 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. The overall specificity of the RT-LAMP test was 99.7% (Wilson’s 95% confidence interval: 98.9 to 99.9%), and the sensitivity for samples with CT \u003c 30 on RT-qPCR was 97.5% (Wilson’s 95% confidence interval: 91.4 to 99.3%) (Fig. 3B and table S2).\n\nMultiplexed sequencing of RT-LAMP reaction products\nOur results indicated that the colorimetric RT-LAMP assay enabled robust identification of positive samples after a 25- to 30-min incubation at 65°C. Validation of positive results, however, required confirmation that the RT-LAMP reaction led to the amplification of viral sequences. To analyze the sequences of many RT-LAMP reaction products, we established multiplexed sequencing of RT-LAMP products (LAMP-sequencing). LAMP-sequencing is based on Tn5 transposase tagmentation (17) and sample barcoding. Tagmentation enables fragmentation and direct adapter ligation of DNA samples for analysis by next-generation sequencing. We used a set of 96 barcoded adapters for tagmentation to barcode the RT-LAMP reaction products in each 96-well plate. After tagmentation, all barcoded fragments from each plate were pooled and size-selected by bead purification to remove excess adapters. A second set of barcoded primers, one per plate-pool, was then used to amplify the tagmented RT-LAMP fragments. 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).\nFig. 4 Multiplexed sequencing of RT-LAMP reaction products (LAMP-sequencing).\n(A) Workflow for LAMP-sequencing is shown. 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. After tagmentation, each plate was pooled individually, followed by removal of excess adapters using size selection. 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). Each dot represents one sample. 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). 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). 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). 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). This indicated that LAMP-sequencing amplified the targeted sequences. 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). 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). For 754 of the 768 samples, we obtained enough reads to make a call (fig. S5). 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 \u003c 30 (Fig. 4B and Table 2). 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.\nTable 2 Summary of LAMP-sequencing results.\nThe 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).\nRT-LAMP\nCT Pos Neg Sum\nLAMP- sequencing Pos RT-qPCR Pos 0–25 49 0 49\n25–30 28 0 28\n30–35 4 0 4\n35–40 0 0 0\nNeg Neg 0 0 0\nNeg RT-qPCR Pos 0–25 0 0 0\n25–30 0 2 2\n30–35 0 16 16\n35–40 0 16 16\nNeg Neg 2 637 639\nToo few reads RT-qPCR Pos 0–25 2 0 2\n25–30 0 0 0\n30–35 0 0 0\n35–40 0 0 0\nNeg Neg 0 12 12\nSum 85 683 768 LAMP-sequencing was performed using the RT-LAMP samples after a prolonged incubation of 40 min at 65°C. At this time point, many of the negative samples and also samples with a CT between 30 and 40 had turned yellow. LAMP-sequencing eliminated all of these samples (Fig. 4C). 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 \u003e 30 min was caused by spurious amplification products and not by late amplification of viral sequences. 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.\n\nA swab–to–RT-LAMP assay without RNA isolation\nRNA isolation is time consuming, costly, and depends on reagents with potentially limited supply during a pandemic. 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.\nSeveral 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. 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. We titrated defined numbers of IVT RNA molecules of the SARS-CoV-2 N gene into swab samples from COVID-19–negative control subjects. 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. S6 and S7, and data file S1). 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.\n\nTesting clinical samples with the swab–to–RT-LAMP assay\nOn 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). As an additional precaution, we kept the samples in the cold (using an ice-cold metal block) whenever possible. For testing large numbers of clinical samples, we performed the RT-LAMP assay using several 96-well plates. 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. 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. This resulted in 235 direct swab–to–RT-LAMP assay measurements and 343 hot swab–to–RT-LAMP assay measurements (Fig. 5A). The hot swab–to–RT-LAMP assay detected a color change in the majority of samples with a CT \u003c 30 with high sensitivity, whereas the direct swab–to–RT-LAMP assay only exhibited a high sensitivity for samples with a CT \u003c 25 (Fig. 5 and Table 3). 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. We found that some positive samples did not induce a color change but did so when assayed a second time. We therefore would recommend running this assay using technical duplicates.\nFig. 5 Swab–to–RT-LAMP assay of clinical pharyngeal swab samples.\n(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). 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). 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). 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).\nTable 3 Shown is RT-qPCR and RT-LAMP testing of 592 clinical samples stratified into CT value bins (see Fig. 5A).\nFig. 5A and table S3 show specificity and sensitivity values calculated from these numbers.\nHot swab–to–RT-LAMP RT-LAMP\nCT Pos Neg Sum\nRT-qPCR Pos 0–25 38 4 42\n25–30 17 5 22\n30–35 5 23 28\n35–40 0 36 36\nNeg Neg 1 214 215\nSum 61 282 343\nDirect swab–to–RT-LAMP RT-LAMP\nCT Pos Neg Sum\nRT-qPCR Pos 0–25 15 1 16\n25–30 6 11 17\n30–35 2 21 23\n35–40 3 23 26\nNeg Neg 9 144 153\nSum 35 200 235\n\nHeterogeneity of specimen pH in the swab–to–RT-LAMP assay\nComparison 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). This was likely due to a sample-specific variability that influenced the starting pH in the LAMP reaction. This might have affected the interpretability of the measurement at 30 min (ΔOD30min). We investigated how this pH shift influenced the RT-LAMP assay. For three plates, the data acquired for the RT-LAMP assay also included measurements for the 10-min time point (ΔOD10min) (Fig. 6A). 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). 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.\nFig. 6 Colorimetric readouts of the swab–to–RT-LAMP assay over time.\n(A) The colorimetric readouts (ΔOD) for the direct (left) and hot (right) swab–to–RT-LAMP assays were assessed every 10 min. 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. Also, the 40-min time point was not available for one plate. The kink in some lines at 30 min (right) was due to a transient equipment malfunction. (B) Comparison of two scoring schemes. 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. The latter shows better separation between positive and negative samples. 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. 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. 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."}

LitCovid-sentences

PMC:7574920 / 9448-32589 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:7574920 / 9448-32589 JSON TXT