In recent times, the need for rapid and sensitive nucleic acid amplification tests (NAATs) to diagnose infectious diseases such as COVID19, tuberculosis etc. has increased. Nucleic acid lateral flow assays (NALFAs) have emerged as a powerful method for detecting amplified products of NAATs in low-resource settings [1]. In practice, the amplified product must be diluted before introducing it into the NALFA to obtain a strong signal. This dilution factor is often determined using experimental trial and error and the rationale behind it is usually not reported. In the realm of lateral flow immunoassays, the idea of hook effect has been well studied, and mathematical models describing it have been developed; according to the hook effect, the test line signal in a lateral flow assay reduces with increasing analyte concentration [2-3], above a threshold analyte concentration. However, this phenomenon has not been well studied for NALFA. In this work, we present a transport reaction model of the NALFA for better fundamental understanding and show that in addition to the hook effect, the presence of unreacted primers in the polymerase chain reaction (PCR) product is also responsible for the reduction in test line intensity; we demonstrate this both experimentally and using the model.

When bi-labelled amplicons are present in excess, it reduces the probability of formation of the full detectable stack at both the test and control lines. Some amplicons completely cover the AuNPs leaving no valency for them to bind to the control line; other amplicons occupy test line antibody sites without any gold nanoparticles bound to them, reducing [BT1] test line signals. Accordingly, dilution of the sample initially increased the test line intensity, and further dilution decreased it; the model showed a similar trend. In the case of presence of unreacted PCR primers, FAM-labeled primers block the reaction sites on the test line, reducing the effective number of binding sites and biotin-labeled primers block the streptavidin sites on AuNPs, reducing the available number of AuNPs for signal generation. Accordingly, as unreacted primers were spiked into the bi-labelled PCR product, the test and control intensities decreased; modelling results showed a similar trend.

These results elucidate that the performance of a NALFA is tightly coupled to the design and conduct of the PCR reaction and that PCR primer concentrations and number of cycles must be carefully chosen for optimal performance of NALFA.