A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture Ligand-Modified...

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• Accurate and rapid genetic testing is crucial for diagnosing infectious diseases and antibiotic resistance.
• Existing methods often require complex laboratory equipment and trained personnel, limiting point-of-care applications.

Purpose of the Study:

• To develop and validate a novel, integrated nucleic acid amplification and detection platform for rapid genetic testing.
• To establish the Rolling Circle Amplification-Lateral Flow Assay (RCA-LFA) for detecting specific DNA targets, including antibiotic resistance genes and viral sequences.

Main Methods:

• Integration of padlock probes, ligation, and rolling circle amplification (RCA) for target DNA amplification.
• Utilized a lateral flow assay (LFA) strip with gold nanoparticles for colorimetric detection of amplified DNA.
• Optimized RCA and LFA parameters, including primer and probe concentrations.

Main Results:

• The RCA-LFA method demonstrated high sensitivity, with a detection limit of approximately 1.3 fmol for the mecA gene.
• The assay exhibited excellent specificity, detecting only the target mecA gene even in the presence of other DNA sequences.
• Successfully applied the RCA-LFA platform for detecting SARS-CoV-2 genes (RdRp and RBD), showcasing its versatility.

Conclusions:

• The developed RCA-LFA method provides a rapid, sensitive, and specific platform for genetic testing.
• This universally applicable method holds significant potential for point-of-care diagnostics of various nucleic acid targets.
• The RCA-LFA assay offers a valuable tool for timely disease diagnosis and antimicrobial resistance surveillance.