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%).

Optimization and Clinical Evaluation of a Multi-Target Loop-Mediated Isothermal Amplification Assay for the Detection of SARS-CoV-2 in Nasopharyngeal Samples

“…With the current conditions, none of the targets were capable of detecting 50 copies. The results obtained are in the same range of other previously published RT-LAMP studies, such as the one from Jang et al, which reached 10 1 -10 2 copies/µL targeting RdRp, E, and N, or that of Mautner et al, who reported limited detection of 10 2 copies/µL [23,32]. Minor differences among assays may be explained due to the usage of different chemicals, primer concentration, and/or amplification temperature and time.…”

“…In this regard, national regulations of different countries highlight the need for multiplex detection of positive result. Jang et al also reported higher sensitivity targeting the N gene in their multiplex RT-LAMP assay implementing strand-displacement probes [32]. Some of the discrepancies between the RT-LAMP and the reference RT-qPCR assays may be explained by the difference in template analyzed (3 vs. 10 µL).…”

“…Regarding the RT-LAMP, out of the 31 positive samples identified, 21 were positive for the three targets, 5 were positive for the combination ORF8-N, and 5 were only positive for N (one of these 5 samples was negative by RT-qPCR and was considered a PD for the later evaluation). Overall, the highest number of positive samples was obtained targeting the N gene, but solely targeting this gene could result in detection problems when thinking on novel variants, which are constantly being reported; thus, targeting more than one gene/fragment would be highly recommended to avoid this issue [32]. In this regard, national regulations of different countries highlight the need for multiplex detection of positive result.…”

Development of multiplex real-time RT-PCR assay for the detection of SARS-CoV-2

“…To achieve this goal, several studies have tackled this challenge and these efforts have yielded several diagnostic kits. Many approaches have been proposed to detect SARS-CoV-2 virus in nasopharyngeal fluids such as multiplex RT-PCR [2,3], CRISPR/Cas12 [4,5], and CRISPR-Cas3 [6], lateral flow immunoassay [7], paper-based biomolecular sensors [8], SHERLOCK testing in one pot [9], DNA aptamer [10], loop-mediated isothermal amplification (LAMP) [11], etc. Each of these methods has its own strong and weak points in terms of sensitivity and specificity.…”

Fast Detection of SARS-CoV-2 RNA Directly from Respiratory Samples Using a Loop-Mediated Isothermal Amplification (LAMP) Test

“…This method can be performed without the use of thermocyclers and tends to be faster than RT-PCR. Several LAMP assays for SARS-CoV-2 detection have been developed [ 4 ], and a limited number of them have been evaluated using clinical samples with promising initial results [ 5 , 6 , 7 , 8 , 9 , 10 ].…”