One-step and Rapid Identification of SARS-CoV-2 using Real-Time Reverse Transcription Loop-Mediated Isothermal...

Area of Science:

• Molecular Biology
• Virology
• Diagnostic Development

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes COVID-19, a significant global health concern.
• Current diagnostic methods like real-time RT-PCR are time-consuming and require specialized equipment.
• There is a need for rapid, portable, and cost-effective diagnostic solutions for widespread disease control.

Purpose of the Study:

• To evaluate a faster, more cost-effective field-based testing method for SARS-CoV-2 detection.
• To develop and validate a simple, rapid screening assay targeting the SARS-CoV-2 Envelope (E) gene using one-step RT-LAMP.
• To establish an alternative diagnostic approach for point-of-care or field use.

Main Methods:

• Total RNA was extracted from respiratory samples of COVID-19 patients.
• A one-step RT-LAMP assay was performed targeting the SARS-CoV-2 E gene.
• LAMP products were visualized via green fluorescence (SYBR Green I), with sensitivity and specificity testing against recombinant plasmids and influenza H1N1.

Main Results:

• The RT-LAMP assay demonstrated high sensitivity, detecting approximately 15 ng of E gene from total RNA and 112 pg from a prepared plasmid.
• The assay exhibited high specificity, distinguishing SARS-CoV-2 from influenza H1N1.
• Results showed RT-LAMP as a potentially viable alternative to real-time RT-PCR for SARS-CoV-2 detection.

Conclusions:

• The developed RT-LAMP assay is a sensitive and specific method for detecting SARS-CoV-2.
• This assay can serve as a portable, rapid, and user-friendly diagnostic tool for field and clinical settings.
• The RT-LAMP method presents a promising alternative to conventional real-time RT-PCR for widespread SARS-CoV-2 screening.