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

Abstract

Since the COVID-19 pandemic outbreak in the world, many countries have searched for quick diagnostic tools to detect the virus. There are many ways to design diagnostic assays; however, each may have its limitations. A quick, sensitive, specific, and simple approach is essential for highly rapidly transmitted infections, such as SARS-CoV-2. This study aimed to develop a rapid and cost-effective diagnostic tool using a one-step Reverse Transcriptase Loop-Mediated Isothermal Amplification (RT-LAMP) approach. The results were observed using the naked eye within 30–60 min using turbidity or colorimetric analysis. The sensitivity, specificity, and lowest limit of detection (LoD) for SARS-CoV-2 RNA against the RT-LAMP assay were assessed. This assay was also verified and validated against commercial quantitative RT-PCR used by health authorities in Saudi Arabia. Furthermore, a quick and direct sampling from the saliva, or buccal cavity, was applied after simple modification, using proteinase K and heating at 98 °C for 5 min to avoid routine RNA extraction. This rapid single-tube diagnostic tool detected COVID-19 with an accuracy rate of 95% for both genes ( ORF1a and N ) and an LoD for the ORF1a and N genes as 39 and 25 copies/reaction, respectively. It can be potentially used as a high-throughput national screening for different respiratory-based infections within the Middle East region, such as the MERS virus or major zoonotic pathogens such as Mycobacterium paratuberculosis and Brucella spp., particularly in remote and rural areas where lab equipment is limited.

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes

“…It does not require RNA isolation but is based on RT-PCR, and it requires an RT-PCR amplification step prior to the strip analysis [16]. Of the RT-LAMP tests, it is worth paying attention to the one prepared by Aldossary et al This test is quick and requires no specialized equipment (only a heat block or water bath to maintain a temperature of 65 • C), and after simple modification, saliva can be used instead of swab material [17]. The disadvantage is that there is no internal control for each test, which affects its reliability.…”

A comprehensive overview on the transmission, pathogenesis, diagnosis, treatment, and prevention of SARS‐CoV‐2

“…The major benefit of RT-LAMP is that it does not need expensive tools or chemicals, which is another benefit. 90 Because this test has a lower cost per reaction than the typical RT-PCR test, Juan and his team were able to demonstrate its affordability. 91 Jonas suggested a new detection method that would be simple to integrate into custom workflow automation.…”

“…This is comparable to improving the RT‐PCR procedure. The major benefit of RT‐LAMP is that it does not need expensive tools or chemicals, which is another benefit 90 . Because this test has a lower cost per reaction than the typical RT‐PCR test, Juan and his team were able to demonstrate its affordability 91 .…”

A colorimetric reverse transcription-loop mediated isothermal amplification (RT-LAMP) method for the rapid detection of SARS-CoV-2 in Thailand

“…Colorimetric RT-LAMP assays for the rapid detection of SARS-CoV-2 RNA have been developed through naked-eye visualization of color changes in the reactions (Lamb et al, 2020;Choi et al, 2023). The methods are versatile and can be broadly adjusted using various approaches (Lamb et al, 2020;Aldossary et al, 2022;Ali et al, 2022). RT-LAMP assays have advantages compared to rapid antigen tests.…”