Development of a paper-based microfluidic assay based on the nucleic acid amplification test (NAAT) for ginseng species authentication

This study aimed to develop a molecular test to differentiate between Panax ginseng (P. ginseng) and Panax quinquefolius (P. quinquefolius), considering their distinct medicinal properties, such as fatigue reduction, brain function improvement, anti-obesity and anti-diabetic potentials, and different market values. A specific locus containing a single nucleotide polymorphism (SNP) site was selected as the discriminating factor. The approach involved employing the lesion-induced DNA amplification (LIDA) isothermal nucleic acid amplification test (NAAT) on a nucleic acid lateral flow assay (NALFA) platform. A NALFA with a colorimetric detection system using gold nanoparticles generated a visible red colour signal upon the presence of target DNA, detectable by the naked eye. Capture probes, designed based on the DNA sequence in the dammarendiol-II synthase (DS) gene of ginseng, were tailored to the SNP site for P. ginseng/P. quinquefolius sequences, while the detection probe was nanogold-labelled. By amplifying and detecting specific SNPs in plant genomic samples, this method demonstrated enhanced specificity, outperforming previous studies and achieving a 30% increase (from 3 to 4.1 and from 2.1 to 2.8) in assay specificity for both P. ginseng and P. quinquefolius, respectively. This improvement was further supported by adjusting the evaporation temperature from room temperature to 37°C temperature, which positively impacted the specificity ratios and improved the selectivity and accuracy in identifying the target samples. Additionally, an evaporation time of 40 minutes was found to be optimal for attaining the highest specificity ratio.