A Simple, Affordable, Rapid, Stabilized, Colorimetric, Versatile RT-LAMP Assay to Detect SARS-CoV-2

Abstract

The SARS-CoV-2 pandemic has forced all countries worldwide to rapidly develop and implement widespread testing to control and manage the Coronavirus Disease 2019 (COVID-19). reverse-transcription (RT)-qPCR is the gold standard molecular diagnostic method for COVID-19, mostly in automated testing platforms. These systems are accurate and effective, but also costly, time-consuming, high-technological, infrastructure-dependent, and currently suffer from commercial reagent supply shortages. The reverse-transcription loop-mediated isothermal amplification (RT-LAMP) can be used as an alternative testing method. Here, we present a novel versatile (real-time and colorimetric) RT-LAMP for the simple (one-step), affordable (~1.7 €/sample), and rapid detection of SARS-CoV-2 targeting both ORF1ab and N genes of the novel virus genome. We demonstrate the assay on RT-qPCR-positive clinical samples, obtaining most positive results under 25 min. In addition, a novel 30-min one-step drying protocol has been developed to stabilize the RT-LAMP reaction mixtures, allowing them to be stored at room temperature functionally for up to two months, as predicted by the Q10. This Dry-RT-LAMP methodology is suitable for potentially ready-to-use COVID-19 diagnosis. After further testing and validation, it could be easily applied both in developed and in low-income countries yielding rapid and reliable results.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

“…Single laboratories using different platforms and targets in SARS-CoV-2 molecular testing can represent a potential variability on Ct values (Rhoads et al, 2020). Considering previous convergent reports and presuming different targets and platforms, the data from the literature show that with an RT-qPCR Ct 30 cutoff, RT-LAMP sensitivity for SARS-CoV-2 detection is close to 100% (Schermer et al, 2020;Thi et al, 2020;de Oliveira Coelho et al, 2021;García-Bernalt Diego et al, 2021) and eventually with a higher threshold Ct 35 as well (L'Helgouach et al, 2020;de Oliveira Coelho et al, 2021). Indeed, we confirm that up to Ct 30 RT-LAMP returned 100% sensitivity for SARS-CoV-2 detection, reaching 98 and 94% when considering Ct values up to 32 and 34, respectively.…”

Detection of SARS-CoV-2 RNA in Urine by RT-LAMP: A Very Rare Finding

“…Recently, we developed an RT-LAMP test for SARS-CoV-2 RNA detection in clinical nasopharyngeal swabs specimens by targeting gene N with a specific-sequence primer set N15 (N15-RT-LAMP) that can be performed as a single-tube colorimetric method, in a real-time platform, and as dry-LAMP. This assay proved to be specific for SARS-CoV-2 and showed a limit of detection of 200 copies per reaction (cpr) [ 27 ]. The RT-LAMP methodology has already been tested successfully in the detection of SARS-CoV-2 RNA in urine but only using artificially spiked samples with various concentrations of SARS-CoV-2 RNA [ 28 ].…”

Development of loop-mediated isothermal amplification (LAMP) assay using SYBR safe and gold-nanoparticle probe for detection of Leishmania in HIV patients

“…Contrastingly, LAMP requires basic equipment, whereas local trained health care workers could perform the assay according to its simple operating procedure. To overcome the limitations of LAMP in the field sites, simplified methods have been successfully modified and adapted to the assays such as selective colorimetric and fluorescent dyes 11 , 13 , 23 , 44 , lyophilized form 45 , paper-based devices 46 and dye capsules 15 , 18 . In the meantime, ongoing development of the new approaches encourages the use of LAMP for point-of-care (POC) in low resource settings such as health services as well as field sites; not only for screening but also to confirm diagnostic level.…”