Abstract
Objectives: Development and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS-CoV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.Methods: Primers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.Results: The analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution was performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.Conclusions: We present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.
Highlights
A new virus causing pneumonia-like infection, COVID-19, which was found in Wuhan, Hubei Province, China, has caused a serious crisis worldwide (Ma et al, 2020)
Analytical sensitivity yielded reliable limit of detection (LOD) of 500 copies/ml
One asymptomatic patient tested positive by qRT-PCR (Ct values 37) and by our RT-LAMP was categorized suspected by in-charge physician and few days later became positive
Summary
A new virus causing pneumonia-like infection, COVID-19, which was found in Wuhan, Hubei Province, China, has caused a serious crisis worldwide (Ma et al, 2020). As of May 24, 2020, COVID-19 cases have been found in 213 countries/regions and infected 5,204,508 patients, 337,687 of whom died (World Health Organization, Situation Report 125). The virulent nature of this virus and its high rate of transmissibility warrants robust, rapid, sensitive, specific, and quantitative diagnostic tools to supplement clinical symptoms aiding clinicians to confidently rule in and rule out patients. Most recently a newer generation of single step RT-LAMP tests were developed to detect SARS-CoV-2, but these assays were not validated with real clinical samples obtained from COVID-19 positive patients (Lamb et al, 2020; Park et al, 2020). Our assay was not developed to be quantitative, our assay was proved to be a rapid and reliable diagnostic tool that potentially could be deployed for high-throughput screening applications in referral and local laboratories
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