Development of a Nucleic Acid Lateral Flow Assay as a Rapid Point of Care Alternative to PCR-Based Diagnostics.

Introduction

Polymerase chain reaction (PCR) remains the gold standard for molecular diagnostics; however, its dependence on specialized thermocycling instrumentation, trained personnel, and extended turnaround times limit its utility in point-of-care and resource-constrained settings. Nucleic acid lateral flow (NALF) assays present a compelling alternative, combining the molecular specificity of nucleic acid amplification with the simplicity and speed of lateral flow detection. This study describes the development and benchmarking of an in-house NALF assay against commercial formats by the Lateral Flow Assay Development Services group at Merck KGaA, Darmstadt, Germany.

Methods

Dual-labelled amplicons (FITC-Biotin, DIG-Biotin, FITC-DIG) generated by PCR were used to evaluate commercial lateral flow tests. An in-house NALF strip was subsequently designed and prototyped on Hi-Flow™ Plus nitrocellulose membrane, incorporating nine strip configurations varying across three streptavidin capture reagents and three anti-FITC detector antibodies conjugated to Estapor® Red Intense Microspheres. Performance was assessed by signal intensity, LOD, and inter-strip precision. Primer spiking experiments were performed to characterize matrix effects arising from residual amplification components.

Results

The in-house NALF assay detected 0.26 fmol per strip for FITC-Biotin amplicons, an improvement over the 1.4 fmol per strip detected by commercial tests. Optimal performance was achieved using streptavidin capture paired with monoclonal anti-FITC antibody conjugated to Estapor® Red Intense Microspheres. The novel High Intense Microspheres delivered enhanced signal intensity over conventional particles, enabling a quicker and more discernible instrument-free visual readout. Primer-spiking experiments revealed up to 41% signal suppression at 0.3 µM free primer, informing purification requirements for downstream integration.

Conclusions

An in-house NALF assay was successfully developed with improved sensitivity over commercial tests, offering instrument-free readout, reduced time-to-result, and point-of-care suitability. Estapor® detection particle sizes support assay tailoring, while colour diversity enables multiplexing capability. The optimised configuration provides a scalable foundation for integration with isothermal amplification, eliminating thermocycling requirements and enabling fully portable nucleic acid diagnostics.