Inner primer Blockers inhibit non-specific amplification in LAMP

The frequent occurrence of non-specific amplification (NSA) in Loop-mediated Isothermal Amplification (LAMP) has significantly constrained its widespread adoption. When the amount of template is limited or absent, NSA products can be randomly generated, leading to unreliable and inaccurate detection results. The mechanisms underlying NSA remain poorly elucidated, resulting in mitigation strategies that inadequately address the fundamental causes and are therefore suboptimal.

Non-template (primers-only) real-time LAMP assays were conducted with three published and three routinely designed primer sets. All reactions demonstrated detectable NSA, thereby supporting the hypothesis that primers constitute a principal source of NSA. Comprehensive analyses of two representative primer sets included single-primer reactions and various multi-primer combinations (ranging from two to six primers) under non-template conditions, a process referred to as primer "SCAN" analysis. These systematic investigations consistently revealed the existence of at least one initiator inner primer (IIP) within each set that was capable of independently initiating NSA. The IIP was consistently identified in the most efficient NSA-amplifying combinations and acted as the primary driver of unwanted amplification events. Importantly, the introduction of unamplified oligonucleotide Blockers specifically targeting F1c/B1c sequences of the IIP significantly inhibited NSA while preserving the original binding specificity, thereby demonstrating the effectiveness and selectivity of targeted inhibition strategies. Adding only one or two Blockers could significantly inhibit NSA. The -3 and -6 shortened Blocker achieve an optimal trade-off between inhibiting NSA and preserving the efficiency of the desired reaction via extending the NSA-free window. In some -6 Blocker groups, the limit of detection (LOD) was improved, greatly enhancing the accuracy of detecting low viral load samples in early stages of infection.

This study confirms that certain primers, particularly IIP, play a key role in initiating NSA in LAMP. Blockers have the potential to serve as a universal strategy to effectively inhibit NSA in LAMP diagnostics. By postpone the times to positive (TTP) of primer-initiated NSA, the probabilities of false positives during a conventional detection time window (30-60 min) are reduced. In particular, some Blockers at suitable length achieved this at only minor cost of sensitivity. Blocker-LAMP approach represent a targeted strategy different from existing general additives or amplicon discrimination ones. It can be applied individually or jointly with other methods to mitigate NSA problem and improve the overall performance of LAMP assays.