Inner primer Blockers inhibit non-specific amplification in LAMP

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostic Assays

Background:

• Non-specific amplification (NSA) frequently occurs in Loop-mediated Isothermal Amplification (LAMP), hindering its diagnostic applications.
• NSA leads to unreliable results, especially with limited or absent template DNA, due to random product generation.
• The underlying mechanisms of NSA are not fully understood, limiting the effectiveness of current mitigation strategies.

Purpose of the Study:

• To investigate the role of primers in initiating NSA during LAMP.
• To identify specific primer components responsible for NSA.
• To develop and evaluate a targeted strategy for inhibiting NSA in LAMP assays.

Main Methods:

• Real-time LAMP assays were performed using various primer sets under non-template conditions.
• Primer "SCAN" analysis was employed, including single-primer reactions and multi-primer combinations.
• Oligonucleotide Blockers were designed to target specific sequences of identified NSA-initiating primers.

Main Results:

• All tested primer sets generated detectable NSA, confirming primers as a primary source.
• An initiator inner primer (IIP) was consistently identified as capable of independently initiating NSA.
• The addition of Blockers targeting IIP sequences significantly inhibited NSA without compromising specificity.
• Optimized Blocker lengths (-3 and -6) balanced NSA inhibition with reaction efficiency, improving the limit of detection (LOD) in some cases.

Conclusions:

• Certain primers, especially IIP, are key initiators of NSA in LAMP.
• Blockers offer a universal and targeted strategy to inhibit NSA in LAMP diagnostics.
• The Blocker-LAMP approach reduces false positives by delaying NSA and can be combined with other methods to enhance assay performance.