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

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

Avicenna J Med Biotechnol. 2024 Jan-Mar;16(1):3-8. doi: 10.18502/ajmb.v16i1.14165.

Authors

Mohammad Shoushtari¹, Mehdi Zeinoddini², Javad Fathi³, Hani Keshavarz Alikhani⁴, Fatemeh Shiekhi²

Affiliations

¹ Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
² Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran.
³ Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
⁴ Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.

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.

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