Development of a loop-mediated isothermal amplification (LAMP)-based electrochemical test for rapid detection of SARS-CoV-2

Abstract

Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/μL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity and 93.33% sensitivity. Our assay provides a rapid, specific, and sensitive test with an electrochemical readout in less than 45 min that could be adapted for point-of-care settings.

Keywords: Devices; Diagnostic technique in health technology.

Graphical abstract

Figure 1

Development of SARS-CoV-2 fluorescent LAMP (A) Schematic of fluorescent RT-LAMP detection. Six LAMP primers comprising outer F3/B3, looping inner FIP/BIP and loop primers LF/LB were designed to target M gene of SARS-CoV-2. Primers, Bst polymerase, RT reverse transcriptase and isothermal reaction buffer were added together to set up the LAMP reaction. RNA was added as the template to the LAMP reaction and the amplification was carried out isothermally at 65°C for 30 min in a Genie II instrument as shown in the scheme. Real time fluorescence was monitored over 30 min. (B) SARS-CoV-2 M gene RNA detection with fluorescent LAMP. Values are mean ± Standard error of the mean (SEM) for both positive (1ng RNA templates) and blank water (n = 4). (C) Cross specificity test for M gene LAMP. Positive panel includes DNA and RNA from 21 respiratory pathogens. Negative panel includes inactivated DNA and RNA from the same 21 respiratory pathogens. Values are mean ± SEM (n = 4). (D) LOD analysis for M gene fluorescent LAMP. Values are mean ± SEM (n = 4). Figure reference Biorender.com; OptiGene.

Figure 2

Development of SARS-CoV-2 electrochemical-RDT (A) Step-by-step procedure for electrochemical LAMP. RT-LAMP isothermal amplification is the first step conducted either in a Genie II instrument or in a heat block. Pyrophosphates generated from the LAMP reactions are cleaved to phosphate ions (PO³⁻) by pyrophosphatase used in the LAMP reaction. The second step involves mixing of LAMP reaction with sodium molybdate, incubated and dropped onto an SPE CNT electrode for CV analysis. A positive LAMP reaction will generate pyrophosphates which ultimately forms phosphomolybdate precipitate that undergoes reduction-oxidation (REDOX) reaction generating electric current (signal). (B) CV showing current profile for a positive reaction conducted with 1ng synthetic SARS-CoV2 RNA as the template and negative with water as the blank reaction (water blank_G). (C and D) Limit of detection (LOD) analysis of the electrochemical LAMP. A varying amount of RNA templates from 0 to 0.1ag were used as the template. CV analysis shows the peaks from positive reactions. Reactions with 100 and 10ag of RNA produced significantly higher currents (μA) than the negative water blank indicating the sensitivity of the electrochemical LAMP as 10ag. Values are mean ± SEM (n = 3–5). ∗ indicate statistical significance between different groups tested using unpaired t-test with Welch’s correction assuming unequal variances; p < 0.05.

Figure 3

Development of electrochemical-RDT for simulated biological samples (A–D) Effect of phosphate removal on electrochemical detection of human serum and saliva samples. There was a significant reduction of background interference in neat serum and saliva samples after phosphate removal step. Values are mean ± SEM (n = 3–4). Sensitivity analysis of SARS-CoV-2 electrochemical LAMP in (B) lysis buffer VTM, (C) human serum and (D) human saliva. Neat serum and saliva were treated with phosphate removal columns to remove any background or matrix phosphates prior to LAMP and electrochemical detection. 10ag is the LOD for the electrochemical LAMP and templates 1fg, 100ag and 10ag were clearly distinguishable from blank samples. The analyses were repeated independently at least 3–4 times for the three categories of simulated samples. (E) Electrochemical detection of KP LAMP in neat and phosphate removed serum. Values are mean ± SEM (n = 3–4). (F) Sensitivity analysis of KP LAMP. Live KP with different dilutions were spiked into phosphate removed human serum. Our test was sensitive to detecting as low as 10 colony forming units. Statistical was done using one-way ANOVA and Tukey’s and unpaired t-test; ∗, p < 0.05; ∗∗, p < 0.005.

Figure 4

SARS-CoV-2 detection in clinical samples using electrochemical-RDT (A) Electrochemical-RDT based detection of positive (red) and negative (blank) samples. 30 clinical swabs were tested, out of which 14 were positive for SARS-CoV-2 in the electrochemical test. These samples were also positive for RT-qPCR test. (B) Average current (μA) for SARS-CoV-2 RT-qPCR identified positive and negative samples. (C) Fluorescent LAMP detection of positive (red) and negative (blank) samples. 11 out of 30 tested samples were positive for SARS-CoV-2 using fluorescent LAMP. (D) Average fluorescence for SARS-CoV-2 positive and negative samples identified by RT-qPCR. Statistical was done using unpaired t-test with Welch’s correction; ∗∗∗, p < 0.0005; ∗∗∗∗, p < 0.00005. P1-P15 are swab samples tested positive by RT-qPCR (shown in red) and N1-N15 are samples tested negative by RT-qPCR.