Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

Saeki Negishi,Haruka Abe,Yui Igasaki,Jiro Yasuda,Gregory Gromowski,Rokusuke Yoshikawa,Hiroaki Goto

doi:10.1371/journal.pntd.0008855

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step–Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.

Highlights

In December 2019, an outbreak of pneumonia caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China [1,2]
We developed the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable platform equipped with a battery for detection of SARS-CoV-2 in clinical specimens
Since RNA extraction step is one of the main obstacles for rapid diagnosis, we developed the RT-LAMP assay without this step, Direct RT-LAMP

Summary

Introduction

In December 2019, an outbreak of pneumonia caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China [1,2]. Until the day of the announcement, over 4,962,707 confirmed cases of SARS-CoV-2 pneumonia in at least 200 countries of all continents, except Antarctica, had been reported [4]. Several reports described that SARS-CoV-2 may show a longer incubation period (~19 days) and human-to-human transmission in the incubation period, making difficult to prevent the infection from spreading [7]. Development of an on-site, rapid, and sensitive diagnostic assay for SARS-CoV-2 infections is of top priority to provide an appropriate treatment and establish a surveillance system to prevent the spread of COVID-19

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Results

Discussion

Conclusion