Rapid visual detection of SARS-CoV-2 by colorimetric loop-mediated isothermal amplification

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• Accurate and rapid detection of SARS-CoV-2 RNA is crucial for controlling the pandemic.
• Existing molecular diagnostic methods can be time-consuming and require specialized equipment.
• Loop-mediated isothermal amplification (LIA) offers a promising alternative for rapid nucleic acid detection.

Purpose of the Study:

• To evaluate a new set of primers targeting the ORF1ab sequence for SARS-CoV-2 RNA detection using LIA.
• To assess the performance of a combined primer set (ORF1ab and N gene) for enhanced sensitivity.
• To determine the feasibility of using the developed protocol for point-of-care diagnostics.

Main Methods:

• Development and testing of novel primer sets targeting SARS-CoV-2 ORF1ab and N gene sequences.
• Optimization of the loop-mediated isothermal amplification (LIA) protocol for visual detection.
• Evaluation of detection limits and specificity using viral RNA standards and clinical samples (saliva).

Main Results:

• The ORF1ab primer set achieved visual detection of SARS-CoV-2 RNA in 16 minutes.
• A combined primer set (ORF1ab + N) significantly increased detection sensitivity, identifying as few as 250 viral RNA copies.
• The protocol successfully detected viral RNA in saliva samples, demonstrating its applicability to easily accessible clinical specimens.

Conclusions:

• The developed LIA assay provides a rapid and sensitive method for SARS-CoV-2 RNA detection.
• The combined primer strategy enhances diagnostic performance, particularly for low viral loads.
• This protocol is suitable for implementation in rapid, point-of-care devices for visual SARS-CoV-2 RNA diagnosis.