A rapid, specific, extraction-less, and cost-effective RT-LAMP test for the detection of SARS-CoV-2 in clinical specimens

Abstract

In 2019 a newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread rapidly from the epicenter in Wuhan (China) to more than 150 countries around the world, causing the Coronavirus disease 2019 (COVID-19) pandemic. In this study, we describe an extraction-less method based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) intended for the rapid qualitative detection of nucleic acid from SARS-CoV-2 in upper respiratory specimens, including oropharyngeal and nasopharyngeal swabs, anterior nasal and mid-turbinate nasal swabs, nasopharyngeal washes/aspirates or nasal aspirates as well as bronchoalveolar lavage (BAL) from individuals suspected of COVID-19 by their healthcare provider. The assay’s performance was evaluated and compared to an RT quantitative PCR-based assay (FDA-approved). With high sensitivity, specificity, and bypassing the need for RNA extraction, the RT-LAMP Rapid Detection assay is a valuable and fast test for an accurate and rapid RNA detection of the SARS-CoV-2 virus and potentially other pathogens. Additionally, the versatility of this test allows its application in virtually every laboratory setting and remote location where access to expensive laboratory equipment is a limiting factor for testing during pandemic crises.

A rapid RT-LAMP SARS-CoV-2 screening assay for collapsing asymptomatic COVID-19 transmission

“…Primer sets match the SARS-CoV2 Ref Seq reference genome 100% (Wuhan-Hu-1; NC 045512.1 ). In-Silico inclusivity analysis by Marino et al, [ 7 ] demonstrated primer sets to differ by one or less mutation in 99.8%, 99.8% and 99.6% (Orf1, E, and N gene respectively against 5773195 SARS-CoV-2 GISAID deposited sequences), with extremely low potential for poor primer hybridization to occur across all three primer sets. Furthermore, minimal In-Silico and wet testing cross-reactivity was observed for pathogens similar or related to SARS [ 7 ].…”

“…During this period, a single repeat swab was requested due to inefficient sampling as identified by failure of amplification within the ACTB control reaction indicating absence of total RNA. In total, 9 RT-PCR confirmed RT-LAMP positive results from a total of 1,673 tests demonstrated a prevalence of asymptomatic infection of 0.54% (540 [7] demonstrated primer sets to differ by one or less mutation in 99.8%, 99.8% and 99.6% (Orf1, E, and N gene respectively against 5773195 SARS-CoV-2 GISAID deposited sequences), with extremely low potential for poor primer hybridization to occur across all three primer sets. Furthermore, minimal In-Silico and wet testing cross-reactivity was observed for pathogens similar or related to SARS [7].…”

“…In total, 9 RT-PCR confirmed RT-LAMP positive results from a total of 1,673 tests demonstrated a prevalence of asymptomatic infection of 0.54% (540 [7] demonstrated primer sets to differ by one or less mutation in 99.8%, 99.8% and 99.6% (Orf1, E, and N gene respectively against 5773195 SARS-CoV-2 GISAID deposited sequences), with extremely low potential for poor primer hybridization to occur across all three primer sets. Furthermore, minimal In-Silico and wet testing cross-reactivity was observed for pathogens similar or related to SARS [7]. Equivalent sensitivity was observed for genomic (Orf1a) and dual sub-genomic (N+E) targets with assays capable of reproducibly detecting 500 copies of Twist Bioscience synthetic positive control RNA.…”

“…New England Biolabs (NEB) developed a dual sub-genomic assay targeting regions of the N and E gene, plus a separate internal control assay targeting the human beta actin gene (ACTB) for confirmation of total RNA indicative of appropriate sample collection [ 6 ]. Marino et al, [ 7 ] went on to multiplex the Orf1a-HMSe, N and E primer sets (plus a primer set targeting 18S RNA), developing an extraction-less Prime CovidDetect ™ Rapid Detection kit. Fowler et al, [ 8 ] optimised and validated OptiGene’s COVID-19 RT-LAMP workflow, successfully establishing the first CE-IVD registered RT-LAMP kit, implemented nationally across several NHS Trusts.…”

A quantitative RT‐qLAMP for the detection of SARS‐CoV‐2 and human gene in clinical application

“…The duration of amplification requires 30 min or less. The LAMP assay for SARS‐CoV‐2 has been widely used in POCT (Anurup et al, 2020 ; Garcia‐Venzor et al, 2021 ; Huang et al, 2022 ; Marino et al, 2022 ; Wei et al, 2021 ). Six primers in the LAMP assay targeting eight distinct sites of the sequence ensure sensitivity and specificity (Hardinge & Murray, 2019 ; Jiang et al, 2020 ; Wang et al, 2015 ).…”