LitCovid-sentences  

PMC:7574920 / 11871-17162 JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T76 0-64 Sentence denotes Testing clinical RNA samples with the colorimetric RT-LAMP assay
T77 65-192 Sentence denotes To evaluate the colorimetric RT-LAMP assay, we needed to compare its sensitivity and specificity to a validated RT-qPCR method.
T78 193-317 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 318-449 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 450-726 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 727-978 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 979-1239 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 1240-1340 Sentence denotes Fig. 2 Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.
T84 1341-1453 Sentence denotes RNA samples isolated from 95 pharyngeal swab specimens were analyzed by the RT-LAMP assay using a 96-well plate.
T85 1454-1683 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 1684-1926 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 1927-2094 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 2095-2153 Sentence denotes Yellow (positive) samples yield a ΔOD of about 0.3 to 0.4.
T89 2154-2186 Sentence denotes Each line represents one sample.
T90 2187-2423 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 2424-2454 Sentence denotes Each dot is one sample (well).
T92 2455-2603 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 2604-2781 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 2782-2900 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 2901-3192 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 3193-3299 Sentence denotes This suggested a detection limit of the colorimetric RT-LAMP assay corresponding to a CT ≈ 30 for RT-qPCR.
T97 3300-3495 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 3496-3579 Sentence denotes The performance of each RT-qPCR run was validated using this as a positive control.
T99 3580-3820 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 3821-3997 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 3998-4292 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 4293-4440 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 4441-4589 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 4590-4713 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 4714-4852 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 4853-5120 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 5121-5291 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.