Rapid and Sensitive Diagnosis of COVID-19 Using an Electricity-Free Self-Testing System

Abstract

Rapid and sensitive detection of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for early diagnosis and effective treatment. Nucleic acid testing has been considered the gold standard method for the diagnosis of COVID-19 for its high sensitivity and specificity. However, the polymerase chain reaction (PCR)-based method in the central lab requires expensive equipment and well-trained personnel, which makes it difficult to be used in resource-limited settings. It highlights the need for a sensitive and simple assay that allows potential patients to detect SARS-CoV-2 by themselves. Here, we developed an electricity-free self-testing system based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) that allows for rapid and accurate detection of SARS-CoV-2. Our system employs a heating bag as the heat source, and a 3D-printed box filled with phase change material (PCM) that successfully regulates the temperature for the RT-LAMP. The colorimetric method could be completed in 40 min and the results could be read out by the naked eye. A ratiometric measurement for exact readout was also incorporated to improve the detection accuracy of the system. This self-testing system is a promising tool for point-of-care testing (POCT) that enables rapid and sensitive diagnosis of SARS-CoV-2 in the real world and will improve the current COVID-19 screening efforts for control and mitigation of the pandemic.

Keywords: SARS-CoV-2; reverse transcription loop-mediated isothermal amplification; self-testing.

Figure 1

Detection of SARS-CoV-2 with the self-testing system. (A) Workflow of detection of SARS-CoV-2 with the self-testing system. The detection process using this self-testing system includes three steps: RNA preparation, RT-LAMP reaction, and data processing. (B) Schematic of incubation system. The system consists of a foam box, a heating bag, and a 3D-printed box. (C) Photograph of the incubation system.

Figure 2

Temperature control effect of the self-testing system. (A) Simulations of temperature distribution inside the foam box. (B) Simulations of temperature distribution (i) without and (ii) with the paraffin. The left column shows the temperature distribution when the temperature inside the 3-D printed box is at maximum, and the right column shows the temperature distribution at 60 min. (C) Different times in the absence and presence of paraffin for temperature ramping, reaction, and out-of-range. (D) The temperature inside the 3D-printed box with different heating bags.

Figure 3

Feasibility test of the system. (A) Image analysis of the positive and negative samples. (B) G/R ratio of negative and positive samples calculated from images taken with a cellphone. The statistical analyses were performed using t-test, two-tailed, where ns = not significant with p > 0.05, the ** denotes significant differences with p values between 0.0001 to 0.001. (C) The images before and after G/R processing. (D) Real-time amplification curve of RT-LAMP with different concentrations of Bst3.0.

Figure 4

Sensitivity evaluation of the self-testing system. (A) Visual outcome of a 10 times dilution of SARS-CoV-2 RNA positive control ranging from 500 to 5 copies/μL. The top image is the original image taken with a cellphone. The bottom image is the binary image after G/R processing. (B) G/R ratio of the RT-LAMP reaction with different concentrations of SARS-CoV-2 RNA. G/R ratio of each concentration was calculated from three parallel tests and represent the mean value of the three tests. The statistical analyses were performed using a one-way analysis of variance, where ns = not significant with p > 0.05, and the ** denotes significant differences with p values between 0.0001 to 0.001.

Figure 5

Test protocol and the design of the self-testing system for POCT. (A) The self-testing system scheme for performing the detection of SARS-CoV-2. (B) Illustrations of the self-testing system. (C) Explored view of the self-testing system. (D) The results of the RNA sample extracted from the SARS-CoV-2 transfected cells by using the self-testing system.