Clinical Validation of a Colorimetric Loop-Mediated Isothermal Amplification Using a Portable Device for the Rapid Detection of SARS-CoV-2

Abstract

Quick and reliable mass testing of infected people is an effective tool for the contingency of SARS-CoV-2. During the COVID-19 pandemic, Point-of-Care (POC) tests using Loop-Mediated Isothermal Amplification (LAMP) arose as a useful diagnostic tool. LAMP tests are a robust and fast alternative to Polymerase Chain Reaction (PCR), and their isothermal property allows easy incorporation into POC platforms. The main drawback of using colorimetric LAMP is the reported short-term stability of the pre-mixed reagents, as well as the relatively high rate of false-positive results. Also, low-magnitude amplification can produce a subtle color change, making it difficult to discern a positive reaction. This paper presents Hilab Molecular, a portable device that uses the Internet of Things and Artificial Intelligence to pre-analyze colorimetric data. In addition, we established manufacturing procedures to increase the stability of colorimetric RT-LAMP tests. We show that ready-to-use reactions can be stored for up to 120 days at -20 °C. Furthermore, we validated both the Hilab Molecular device and the Hilab RT-LAMP test for SARS-CoV-2 using 581 patient samples without any purification steps. We achieved a sensitivity of 92.93% and specificity of 99.42% (samples with CT ≤ 30) when compared to RT-qPCR.

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

Figure 1

The Hilab Molecular system. (a) From swab collection to result in a timeline, describing the integrated system workflow. Samples are first submitted to a heat process for viral inactivation and then pipetted into the reaction tubes. The whole test can be monitored in real-time and the diagnosis is given by a healthcare professional assisted by artificial intelligence. Patients receive the results automatically by SMS or email in less than 1 h. (b) The molecular Hilab device. Each SARS-CoV-2 test cartridge is composed of negative control (1), internal control (2), test tube (3), and positive control (4). The colorimetric measurements are performed using an LED at the top and a colorimetric sensor under the tubes. (c) Schematic representation of the SARS-CoV-2 genome, with detailed information about the primers sites for genes E and N.

Figure 2

Colorimetric LAMP data. After 30 min of incubation, two images are generated: (a) is the first image which consists of the color change of the tube reactions during the 30 min, and the second image (b) is a curve with the delta of the color change. In graph (b), a different color represents each tube. The negative control tube is represented in red, the internal control tube in orange, the test tube in green, and the positive control in blue. The X-axis represents the time of the reaction in seconds. These images are sent for analysis by a healthcare professional with the anamnesis questionnaire.

Figure 3

The Hilab Molecular sensor diagram.

Figure 4

Comparison between the CT (cycle threshold) obtained from RT-qPCR for the gene N and the Cy0 (seconds) from RT-LAMP. The colors represent the qualitative result of RT-LAMP. Positive samples in RT-LAMP (Cy0 < 1200 s) are displayed as green dots and negative samples as red dots.

Figure 5

Cross-reaction experiment. SARS-CoV-2 primers were tested against Influenza A (1), Influenza B (2), and RSV (3). Images were captured before (I) and after (II) the reaction. Positive (+C) and negative (−C) controls are shown.

Figure 6

Colorimetric RT-LAMP long-term stability. In 30-day intervals, the stability of the reaction was tested in 2 replicates (A and B), with photos of the tubes right after the thawing of the reagents (Upper lane) and also at the end of the reaction (Lower lane). The corresponding day is given on the left side. The test was composed of a negative control (−C), with no DNA, and a positive control (+C) containing pUC57 plasmids cloned with the sequences for genes E and N of the SARS-CoV-2 genome in equal amounts.