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

Factors that affect the performance of the nucleic acid lateral flow assay (NALFA) have not been well studied. In this work, we identify two important phenomena that negatively affect signal intensities during the detection of PCR products using NALFA: (i) the presence of unreacted PCR primers, and (ii) the presence of excess PCR amplicons. This is the first report that highlights the negative effect of unreacted PCR primers on NALFA. The negative effect of excess amplicons, while not explicitly reported for NALFAs, emanates from an identical phenomenon in lateral flow immunoassays known as the "hook effect". We show that the above effects may be alleviated by increasing the concentration of capture antibodies at the test line and the concentration of reporter moieties (gold nanoparticles). To demonstrate these, we utilized a PCR assay in which both primers were end-labeled, to generate dually end-labeled (bi-labeled) PCR amplicons of 230 bp length. To provide mechanistic understanding of these phenomena, we present the first transport-reaction model of NALFA, the results of which qualitatively matched all observed phenomena. Based on these results, we provide recommendations for the optimal design of PCR for NALFA detection.