Testing clinical RNA samples with the colorimetric RT-LAMP assay
To 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).
Fig. 2  Sensitivity and specificity of the RT-LAMP assay compared to RT-qPCR using clinical samples.
RNA 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 < 30 changed the color of the phenol-red dye within the first 30 min of the reaction. 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). 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 < 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.
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). 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.
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). 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).
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). 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.