Development of Reverse Transcription Loop-Mediated Isothermal Amplification Assays Targeting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Abstract

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.

Figure 1

Loop-mediated isothermal amplification (LAMP) primer positions on aligned sequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV. Primer binding sites of Nsp3_1-61 (A) and Nsp3_2-24 (B) LAMP primer sets are depicted on aligned sequences of five SARS-CoV-2 (from the top, MN908947, MN938384, MN988713, MN985325, and MN975262) and seven SARS-CoV (from the top, NC_004718, AY613947, AY313906, AY559094, AY502924, AY278491, and AY502927). Conserved sites are toggled at 50% level by MEGA software version 7 so that SARS-CoV-2 specific residues versus SARS-CoV are background colored. Different color bars are used as follows to distinguish binding sites: F3, blue; F2, red; F1, orange; loop forward (LF), green; loop backward (LB), light green; B1, light orange; B2, brown; and B3, light blue.

Figure 2

Cross-reactivity to other coronaviruses tested for reverse transcription loop-mediated isothermal amplification (LAMP) assay targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Real-time amplification fluorescence signal and end-point leuco crystal violet colorimetric detection results of cross-reactivity test for Nsp3_1-61 (A) and Nsp3_2-24 (B) LAMP primer sets. Reactions are performed with 20 U/reaction of reverse transcriptase and optimized temperature, dNTP concentration, and Mg²⁺ concentration for each primer set. RNA copy number of each viral RNA is as follows: human coronavirus (hCoV)–229E, 1.6 × 10⁶; hCoV-OC43, 1.6 × 10⁶; Middle East respiratory syndrome coronavirus (MERS-CoV), 4.5 × 10⁶; and SARS-CoV-2, 2.5 × 10³. NTC, no template control.

Supplemental Figure S1

Sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) loop-mediated isothermal amplification (LAMP) assays to cDNA dilutions. Real-time amplification fluorescence signal (A) and end-point leuco crystal violet (LCV) colorimetric detection results (B) of four SARS-CoV-2 LAMP primer sets that were subjected to reaction optimization. Color of amplification signal curve and LCV detection label for each cDNA dilution are matched. Designated copy number is corresponding reverse transcription input RNA copy number. Some low fluorescence of amplification signal plateaus is due to baseline correction process of LightCycler 96 software. NTC, no template control.

Supplemental Figure S2

Colorimetric detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription loop-mediated isothermal amplification limit of detection (LoD) tests for Nsp3_1-61 and Nsp3_2-24. Leuco crystal violet colorimetric detection results of LoD tests for Nsp3_1-16 and Nsp3_2-24. A 20 U/reaction of reverse transcriptase was used. NTC, no template control.