Development of a multiplex Loop-Mediated Isothermal Amplification (LAMP) assay for on-site diagnosis of SARS CoV-2

Abstract

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).

Fig 1. Limit of detection for the monoplex SARS CoV-2 RT-LAMP assay.

The limit of detection for the monoplex SARS CoV-2 RdRP (A), E (B) and N (C) RT-LAMP assay was tested with synthetic RdRP, E and N plasmid ranging from 10⁸ to 10⁰ copies/μL, respectively. Numbers (1–10) indicated plasmid copy numbers/μL (1.0 × 10⁸–1.0 × 10⁰ copies/μL) and negative control (distilled water (DW) as non-template control).

Fig 2. Limit of detection (LOD) tests of the monoplex and multiplex RT-LAMP assay, Allplex™ 2019-nCoV Assay and PowerChek™ 2019-nCoV Real-time PCR kit for clinical SARS CoV-2 NP sample (range of 10⁻³–10⁻⁷).

(A) Detection limits of monoplex SARS CoV-2 RdRP (left), E (middle) and N (right) LAMP primer sets. (B) Detection limits of the multiplex SARS CoV-2 RdRP/E (upper left), RdRP/N (upper middle), E/N (upper right), RdRP/E/N (lower left) and RdRP/N/IC (lower middle) LAMP primer sets (C) Detection limits of Allplex™ 2019-nCoV assay (Left) and PowerChek™ 2019-nCoV Real-time PCR kit (Right).