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%).
Highlights
The truepositive rate was defined as the proportion of SARS-CoV-2 positive which is correctly identified by the multiplex reverse-transcription loopmediated isothermal amplification (RT-Loop-mediated isothermal amplification (LAMP)) assay compared to the AllplexTM
The plasmids were serially diluted 10-fold from 1 × 108 copies/μL to 1 × 100 copies/μL to determine the detection of limit of the multiplex SARS CoV-2 RNA-dependent RNA polymerase (RdRP)/N/IC RT-LAMP assay
The sensitivity of the SARS CoV-2 RdRP, E and N gene RT-LAMP was evaluated by testing synthetic plasmid standards, including synthetic partial RdRP, E and N genes ranging from 108 to 100 copies/μL, respectively (Fig 1)
Summary
In December 2019, an outbreak in Wuhan, China of a severe respiratory illness was caused by a previously unrecognized coronavirus, which has since been named severe acute respiratory. An RT-qPCR-based test distributed by WHO is being deployed in many countries to detect SARS CoV-2 RNA, and several commercial RT-qPCR kits (PowerChekTM 2019-nCoV Real-time PCR kit [Kogenebiotech, Seoul, South Korea], granted EUAL in Korea) are available to diagnose SARS CoV-2 in Korea. These RT-qPCR detection methods require nearly three hours to produce results, and skilled technicians and advanced laboratory infrastructure are necessary. We have developed multiplex SARS CoV-2 LAMP primer/probe sets using strand-displaceable probes, based on the region of the RdRP, E and N gene of the aligned sequences of SARS CoV-2 subtypes. The performance of the multiplex SARS CoV-2 RdRP/N/IC LAMP assay was compared with direct RT-qPCR methods using the Seegene AllplexTM 2019-nCoV Assay and Kogenebiotech PowerChekTM 2019-nCoV Real-time PCR kit for SARS CoV-2 clinical samples
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