Detection of Coronaviruses Using RNA Toehold Switch Sensors

Abstract

A rapid, sensitive and simple point-of-care (POC) nucleic acid diagnostic test is needed to prevent spread of infectious diseases. Paper-based toehold reaction, a recently emerged colorimetric POC nucleic acid diagnostic test, has been widely used for pathogen detection and microbiome profiling. Here, we introduce an amplification method called reverse transcription loop-mediated amplification (RT-LAMP) prior to the toehold reaction and modify it to enable more sensitive and faster colorimetric detection of RNA viruses. We show that incorporating the modified RT-LAMP to the toehold reaction detects as few as 120 copies of coronavirus RNA in 70 min. Cross-reactivity test against other coronaviruses indicates this toehold reaction with the modified RT-LAMP is highly specific to the target RNA. Overall, the paper-based toehold switch sensors with the modified RT-LAMP allow fast, sensitive, specific and colorimetric coronavirus detection.

Keywords: middle east respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), molecular diagnostics; reverse transcription loop-mediated isothermal amplification (RT-LAMP).

Figure 1

Target RNA detection scheme using the modified reverse transcription loop-mediated amplification (RT-LAMP) developed in this study and subsequent toehold reaction in a paper-based cell-free protein expression system. The reporter protein, beta-galactosidase, catalyzed the conversion of chlorophenol red-D-galactopyranoside (CPRG) to chlorophenol red (CPR). Beta-galactosidase structure was from PDB ID 6DRV, beta-galactosidase [17]. (Abbreviation: RBS, ribosome binding site; TL, in vitro translation; TX, in vitro transcription).

Figure 2

Toehold switch sensor screening. (a) Target regions and normalized ensemble defect (%) of each toehold switch sensor were indicated. (b) Absorbance changes at 570 nm of toehold reaction with or without trigger RNA in a cell-free system on a paper-disc. Blue line: toehold reaction with trigger RNA (on-state), Black line: toehold reaction without trigger RNA (off-state). (c) Fold changes of toehold switch sensors in Figure 2b at 60 min. (d) Sensitivity of sensor M6 and S1 with different concentrations of trigger RNA. Fold change was calculated as the toehold reaction with trigger RNA divided by that without trigger at 60 min. Two-tailed student’s test; *** p < 0.001; Error bars represent ± s.d., n = 3.

Figure 3

Schematic illustration of the modified RT-LAMP reaction consists of a starting-structure-producing step (a) and a cycling amplification step (b). (c) T7 promoter (P_T7) and complementary T7 terminator (T_T7) were inserted into inner primers, FIP and BIP, respectively. With different combinations of the modified primers, RT-LAMP and the subsequent toehold reaction in the paper-based cell-free system were performed. Fold change was calculated as the absorbance value with trigger RNA divided by that without the trigger RNA. (d) Complementary T7 promoter (P_T7) and T7 terminator (T_T7) were inserted into inner primers, FIP and BIP, respectively. Sensor M6 and its target RNA (20 pM) were used and the RT-LAMP was done without loop primers. For the fold change calculation, the absorbance value was obtained with 60-min RT-LAMP and 60-min toehold reaction. For the negative control (NC), the RT-LAMP reaction product with no target RNA was used. Two-tailed student’s test; * p < 0.05; Error bars represent ± s.d., n = 3.

Figure 4

Detection limit of the modified RT-LAMP-coupled toehold reaction on contrived samples. The modified RT-LAMP is performed for 60 min with different combinations of loop primers (Loop B and Loop F). The resulting RT-LAMP product was applied to the subsequent toehold reaction. Fold change was calculated as the absorbance value from the toehold reaction with trigger RNA divided by that without the trigger RNA at 60 min. (a) Schematic illustration of the modified RT-LAMP and the subsequent toehold reaction. The paper assay image was the result of Figure 4d for sensor M6. (b) Effect of loop primers to improve detection limit of sensor M6. (c) Effect of different combinations of loop primers to detection limit of sensor S1. (d) Compatibility of human saliva with RT-LAMP-coupled toehold reaction. Loop B and Loop F were used for sensor M6 and S1. Two-tailed student’s test; * p < 0.05, ** p < 0.01, *** p < 0.001; Error bars represent ± s.d., n = 3.

Figure 5

Reduction of the overall reaction time for the modified RT-LAMP and the subsequent toehold reaction. For the reduction of overall reaction time, 120 copies of target RNA for sensor M6 were used. (a) Absorbance (570 nm) changes of the toehold reaction with or without trigger RNA using sensor M6. Blue and green lines indicate the toehold reaction after 20-min and 60-min RT-LAMP reactions, respectively, in the presence of target RNA. The black line denotes the same reaction for 60 min in the absence of target RNA. (b) Fold change was calculated as the absorbance values from toehold reaction with trigger RNA divided by that without the trigger RNA at 40 min. For the negative control (NC), the RT-LAMP reaction product with no target RNA was used. Two-tailed student’s test; * p < 0.05; Error bars represent ± s.d., n = 3.

Figure 6

Specificity of the modified RT-LAMP-coupled toehold reaction against other coronaviruses. Sensor M6 for MERS-CoV and sensor S1 for SARS-CoV-2 were tested with several coronaviral RNAs. For the specificity test, 12,000 copies of target RNA were used for RT-LAMP. Fold change was calculated by taking absorbance values at 60 min using a 60-min RT-LAMP reaction product. Colors in the heatmap indicate the mean value (n = 3) of fold changes of the toehold reaction and the scales are given on the right.