Unreacted Labeled PCR Primers Inhibit the Signal in a Nucleic Acid Lateral Flow Assay as Elucidated by a Transport...

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Assay Development

Background:

• Nucleic acid lateral flow assays (NALFA) are widely used for rapid detection of nucleic acids.
• Factors influencing NALFA performance, particularly signal intensity, require further investigation.

Purpose of the Study:

• To identify and characterize factors negatively impacting signal intensity in NALFA for PCR product detection.
• To investigate the effects of unreacted PCR primers and excess amplicons on NALFA performance.
• To propose strategies for optimizing NALFA signal intensity.

Main Methods:

• Utilized a PCR assay generating dually end-labeled (bi-labeled) PCR amplicons.
• Investigated the impact of varying concentrations of unreacted primers and amplicons.
• Modified capture antibody and reporter moiety (gold nanoparticle) concentrations.
• Developed and applied a transport-reaction model for NALFA.

Main Results:

• Unreacted PCR primers were identified as a novel factor negatively affecting NALFA signal intensity.
• Excess PCR amplicons exhibited a 'hook effect,' reducing signal intensity.
• Increased concentrations of capture antibodies and gold nanoparticles mitigated these negative effects.
• The developed NALFA model qualitatively matched experimental observations.

Conclusions:

• Unreacted primers and excess amplicons are critical factors affecting NALFA performance.
• Optimizing antibody and nanoparticle concentrations can enhance NALFA signal.
• Recommendations for PCR design are provided to improve NALFA detection accuracy.