One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse  Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

Mohammad  Shoushtari; Mehdi  Zeinoddini; Javad  Fathi; Hani Keshavarz  Alikhani; Fatemeh  Shiekhi

doi:10.18502/ajmb.v16i1.14165

Mohammad Shoushtari Department of Virology, Pasteur Institute of Iran, Tehran, Iran
Mehdi Zeinoddini Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran
Javad Fathi Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Hani Keshavarz Alikhani Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Fatemeh Shiekhi Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran

DOI: https://doi.org/10.18502/ajmb.v16i1.14165

Keywords: COVID-19, Detection, LAMP Assay, Real time PCR, SARS-CoV-2

Abstract

Background: SARS-CoV-2 as the cause of novel coronavirus disease (COVID-19) is a member of the family Coronaviridea that has generated an emerging global health concern. Controlling and preventing the spread of the disease requires a simple, portable, and rapid diagnostic method. Today, a standard method for detecting SARS-CoV-2 is quantitative real-time reverse transcription PCR, which is time-consuming and needs an advanced device. The aim of this study was to evaluate a faster and more cost-effective field-based testing method at the point of risk. We utilized a one-step RT-LAMP assay and developed, for the first time, a simple and rapid screening detection assay targeting the Envelope (E) gene, using specific primers.

Methods: For this, the total RNA was extracted from respiratory samples of COVID-19 infected patients and applied to one-step a RT-LAMP reaction. The LAMP products were visualized using green fluorescence (SYBR Green I). Sensitivity testing was conducted using different concentrations of the designed recombinant plasmid (TA-E) as positive control constructs. Additionally, selectivity testing was performed using the influenza H1N1 genome. Finally, the results were compared using with conventional real time RT-PCR.

Results: It was shown that the RT-LAMP assay has a sensitivity of approximately 15 ng for the E gene of SARS-CoV-2 when using extracted total RNA. Additionally, a sensitivity of 112 pg was achieved when using an artificially prepared TA-E plasmid. Accordingly, for the detection of SARS-CoV-2 infection, the RT-LAMP had high sensitivity and specificity and also could be an alternative method for real-time RT-PCR.

Conclusion: Overall, this method can be used as a portable, rapid, and easy method for detecting SARS-CoV-2 in the field and clinical laboratories.