Nucleic acid tests and nucleic acid amplification tests for ginseng species authentication conducted on the microfluidic chip

Panax ginseng and Panax quinquefolius, which are commonly called Chinese/Korean ginseng and Canadian/American ginseng, have different medical properties and market values; however, they can be difficult to differentiate from one another based on physical appearances especially when the samples are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this is based on the nucleic acid test (NAT) conducted on the microfluidic chip. Three single nucleotide polymorphism (SNP) sites on the Panax genome that differ between. P. ginseng and P. quinquefolius have been studied using four different NAT methodologies involving probe hybridization to one of the three SNP sites (N1, N2, N3) on the antisense strands amplified by asymmetric PCR. These NATs are distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand) and by the liquid flow method (i.e. static or dynamic). These methods are probe‒target method, target‒probe method, capture strand‒target‒probe method and dynamic probe‒target method. Several new probe/target design rules for the NATs have been developed from this study. Out of the four methods, it was found that the capture strand-target-probe method provided the best differentiation, in which a 3'-NH2 capture strand is first immobilized, the antisense PCR strand is then bound, while N2G and N3Q probes are finally used for detection of P. ginseng (G) and P. quinquefolius (Q), respectively. One of the three SNP sites (N2) has been selected to differentiate different Panax species using a nucleic acid amplification test (NAAT) known as lesion-induced DNA amplification (LIDA) which has been developed for use in the microfluidic chip. This isothermal NAAT not only amplifies the extracted plant genomic samples but also allows for detection of specific SNPs better than the conventional NAT. This method was used to authenticate 14 ginseng samples (powdery, granular, root); next generation sequencing by Illumina was used to verify the NAAT results.