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

Abstract

SARS-CoV-2 is the coronavirus responsible for COVID-19, which has spread worldwide, affecting more than 200 countries, infecting over 140 million people in one year. The gold standard to identify infected people is RT-qPCR, which is highly sensitive, but needs specialized equipment and trained personnel. The demand for these reagents has caused shortages in certain countries. Isothermal nucleic acid techniques, such as loop-mediated isothermal amplification (LAMP) have emerged as an alternative or as a complement to RT-qPCR. In this study, we developed and evaluated a multi-target RT-LAMP for the detection of SARS-CoV-2. The method was evaluated against an RT-qPCR in 152 clinical nasopharyngeal swab samples. The results obtained indicated that both assays presented a “good concordance” (Cohen’s k of 0.69), the RT-LAMP was highly specific (99%) but had lower sensitivity compared to the gold standard (63.3%). The calculated low sensitivity was associated with samples with very low viral load (RT-qPCR Cq values higher than 35) which may be associated with non-infectious individuals. If an internal Cq threshold below 35 was set, the sensitivity and Cohen’s k increased to 90.9% and 0.92, respectively. The interpretation of the Cohen’s k for this was “very good concordance”. The RT-LAMP is an attractive approach for frequent individual testing in decentralized setups.

Moving towards on-site detection of Shiga toxin-producing Escherichia coli in ready-to-eat leafy greens

“…The LOD50 values obtained were very similar to those previously reported for LAMP, and qPCR-based methods, targeting STEC or other foodborne pathogens (Alejandro Garrido-Maestu et al, 2020 ; Lamas et al, 2023 ; Sohrabi et al, 2022 ; Yu et al, 2020 ). Even though it was not part of the focus of the current study, following the reported methodology improved the LOD of the qPCR assay set as a reference, 8.7 CFU/25 g, as all the samples resulted positive, including those spiked with 1.4 CFU/25 g, in agreement with previous studies which reported higher sensitivity of qPCR-based methods compared to LAMP ( Deguo et al, 2008 ; A Garrido-Maestu et al, 2018 ; Roumani et al, 2021a , 2021b ). It must be noted that the original qPCR study was focused on same-day detection thus was possible to be performed in only 5 h.…”

“…The new stx 1/2 LAMP assays were optimized (simplex assays) taking advantage of fluorescence real-time LAMP, following the procedure described by Roumani et al ( Roumani et al, 2021a , 2021b ), see Supporting Information Figures S1A to S1F and S2A to S2E . The final reaction volume was 25 μL composed of 15 μL of GspSSD2.0 Fast Isothermal Master Mix (ISO-004, OptiGene Ltd., Horsham, UK), 0.04 μL of ROX as a passive Reference Dye (Invitrogen™, Carlsbad, CA, USA), 1% Pullulan (TCI Europe, Zwinjdrecht, Belgium), 1 μL of stx 1 and stx 2 25X primer stock (multiplex, or STEC LAMP assay, see Table 2 for detailed primer concentration) and 5 μL of template DNA, the remaining volume was filled with sterile milliQ water.…”

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

“…Although five primer-probe sets were designed, several results were obtained using two or three of the viral targets detected with FAM-labelled probes, which was equivalent to perform 3- or 2-plex rRT-PCR assays. One alternative to multiplex rRT-PCR approach consists of performing multi-target loop-mediated isothermal amplification (LAMP) [ 50 ]. Although real-time LAMP was specific, this method exhibited lower sensitivity compared to rRT-PCR because none of the LAMP primers were capable of detecting SARS-CoV-2 target genes down to fifty copies on patient samples [ 50 ].…”

“…One alternative to multiplex rRT-PCR approach consists of performing multi-target loop-mediated isothermal amplification (LAMP) [ 50 ]. Although real-time LAMP was specific, this method exhibited lower sensitivity compared to rRT-PCR because none of the LAMP primers were capable of detecting SARS-CoV-2 target genes down to fifty copies on patient samples [ 50 ].…”

Development and Validation of Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple and Rapid Diagnostic Tool for SARS-CoV-2 Detection

“…Roumani et al have also developed and evaluated RT-LAMP for COVID-19 detection compared to RT-qPCR for 152 clinical nasopharyngeal swabs. The finding indicated that both techniques have a good concordance, with the RT-LAMP having a high specificity (99%) and lower sensitivity (63.3%) compared to the RT-qPCR [ 16 ]. Anastasiou et al tested using LAMP to detect SARS-CoV-2 genomes directly in respiratory samples without further extracting the viral nucleic acids beforehand.…”