Rapid and highly sensitive one-tube colorimetric RT-LAMP assay for visual detection of SARS-CoV-2 RNA

Abstract

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly contagious disease. To tame the continuously raging outbreak of COVID-19, developing a cheap, rapid and sensitive testing assay is absolutely imperative. Herein, we developed a one-tube colorimetric RT-LAMP assay for the visual detection of SARS-CoV-2 RNA. The assay integrates Si-OH magnetic beads (MBs)-based fast RNA extraction and rapid isothermal amplification in a single tube, thus bypassing the RNA elution step and directly amplifying on-beads RNA molecules with the visualized results. This one-tube assay has a limit of detection (LOD) as low as 200 copies/mL for sample input volumes of up to 600 μL, and can be performed in less than 1 h from sample collection to result readout. This assay demonstrated a 100% concordance with the gold standard test RT-qPCR test by using 29 clinical specimens and showed high specificity. This one-tube colorimetric RT-LAMP assay can serve as an alternative platform for a rapid and sensitive diagnostic test for COVID-19 and is particularly suitable for use at community clinics or township hospitals.

Keywords: LAMP; One-tube; Point-of-care test; SARS-CoV-2; Visual readout.

Fig. 1

Schematic of the one-tube colorimetric RT-LAMP assay for the visual detection of viral RNA. This assay is developed via the integration of Si–OH MB-based fast viral RNA extraction and rapid isothermal amplification in a single tube in less than 1 h.

Fig. 2

Optimization and characterization of MBs-based nucleic acid extraction and RT-LAMP reaction tolerance to MBs. (A) Effect of MBs size on RNA extraction efficiency, (B) Effect of incubation time on RNA extraction efficiency, (C) Characterization of the extraction yield of MBs using various concentrations of GX/P2V virus particles under different sample volumes, (D) Assessment of RT-LAMP reaction tolerance to MBs by using GX/P2V RNA, the inset picture is corresponding colorimetric RT-LAMP result, P represents positive control using plasmid DNA as template.

Fig. 3

Sensitivity of the one-tube colorimetric RT-LAMP assay for GX/P2V virions and SARS-CoV-2 RNA. (A) Sensitivity test using GX/P2V virus particles. (B) Reproducibility test using 200 copies/mL GX/P2V virus particles with various input volumes and a constant amount of MBs. (C) Reproducibility test using 200 copies/mL GX/P2V virus particles with various input volumes and the proportional increase of MBs amounts. (D and E) Sensitivity test for SARS-CoV-2 using RdRp primer sets with 600-μL input volume. (F) Reproducibility test using 600 μL of the 200 copies/mL SARS-CoV-2 RNA solution. In above tests, negative control (NTC) was performed using 1 × PBS instead of samples.

Fig. 4

Specificity and clinical validation of the one-tube colorimetric RT-LAMP assay using clinical SARS-CoV-2 samples. (A) Specificity test for SARS-CoV-2 detection using RdRp and NSP2 primer sets. GX/P2V RNA and one clinical SARS-CoV-2 RNA were used to test the cross-reactivity of LAMP assay. (B) Sample pooling strategies with sample pool sizes of 2–4. (C) Detection of clinical SARS-CoV-2 RNA using one-tube colorimetric RT-LAMP assay. The inset table shows corresponding Ct values (ORF1ab target gene) of the clinical specimens. (D) The Ct values of the clinical samples targeting three different regions of SARS-CoV-2. Ct values were obtained using an authorized 2019-nCoV RT-PCR Detection Kit (20203400299) with a LOD of 300 copies/mL. A Ct value less than 41 indicates a positive result with the sample denoted as LAMP positive. Ct value greater than 41 indicates a negative result with the samples denoted as LAMP negative.