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.
Highlights
Coronavirus disease 19 (COVID-19) is an infection caused by the novel coronavirus SARS-CoV-2, which emerged in China in December of 2019, becoming the seventh member of the Coronaviridae family to infect humans [1]
With the aim of contributing to an effective COVID-19 diagnosis, we present a novel, specific, sensitive, rapid, and versatile RT-loop-mediated isothermal amplification (LAMP) assay for SARS-CoV-2
Four of the eight primer sets were selected for further evaluation based on the shorter time to positivity (Tp)— meaning the fastest amplification—reproducibility and the absence of non-specific amplifications: set ORF1ab (Tp = 20.5 min), set E (Tp = 43.5 min), set N5 (Tp = 20 min), and set N15 (Tp = 15 min)
Summary
Coronavirus disease 19 (COVID-19) is an infection caused by the novel coronavirus SARS-CoV-2, which emerged in China in December of 2019, becoming the seventh member of the Coronaviridae family to infect humans [1]. It is less severe than other previously described coronaviruses infecting humans, such as SARS or MERS coronaviruses, it has a significantly higher transmission capacity. Diagnosis in the early infection stages is hindered by the aforementioned incubation period together with the asymptomatic course or unspecific manifestations of the illness in a high proportion of patients [5]. One of the main challenges to contain the spread of COVID-19 is the identification of asymptomatic cases
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