Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

Abstract

Background: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results.

Methods: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm.

Results: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high.

Conclusion: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

Keywords: Point of Care; RT-LAMP; SARS-CoV-2.

Fig. 1

Colorimetric RT-LAMP color display. Tube (a) positive (b) intermediate and (c) negative. This analysis was made by naked eye as in a real case scenario

Fig. 2

RT-LAMP validation results. a Electrophoresis of RT-LAMP products. RT-LAMP reaction products were analyzed on a 2% agarose gel and the DNA was stained with ethidium bromide. The typical band pattern of a RT-LAMP reaction was visible in positive samples that showed a color change from pink to yellow. Lane 1 = NTC and lanes 2–6 = positive samples. b Colorimetric readout after 30 min of RT-LAMP reaction. After the reaction, the optical densities were measured at 434 nm and 560 nm using a BioteK Synergy reader. To obtain the ΔOD (color change difference), the absorbance at 560 nm was subtracted from the one at 434 nm. The line inside the box indicates the median and the whiskers extend either to the minimum and maximum values. * Indicates difference (p > 0.001) between groups (unpaired, t-test). c Receiver Operating Characteristic (ROC) curve for SARS-CoV-2 colorimetric RT-LAMP with area under the curve of 0.92 (p < 0.0001) and 95% confidence interval (CI) 0.8924 to 0.9483. d Detection of SARS-CoV-2 through colorimetric RT-LAMP assay and comparison between colorimetric RT-LAMP results and RT-qPCR Ct values. Between the dotted lines is a conflicted area where most of the intermediate samples are found

Fig. 3

RT-LAMP internal control results. a Colorimetric readout after 30 min of 18S internal control RT-LAMP reaction. Samples were pipette in a 384-well plate and the optical density was measured at 434 nm and 560 nm using a BioteK Synergy reader. To obtain the ΔOD (color change difference), absorbance at 560 nm was subtracted from the one at 434 nm. The line inside the box indicates the median and the whiskers extend either to the minimum and maximum values. * Indicates difference (p > 0.001) between groups (unpaired, t-test). b Detection of internal control 18S by RT-LAMP assay. Comparison between colorimetric RT-LAMP results and RT-qPCR CT values (RNAse P)