3.4. The Validation of adulterant ingredient, Panax quinquefolius, found in pharmaceutical samples based on SNP-detection method
One OTU has been assigned to be P. quinquefolius in the pharmaceutical sample HSZY192 by the shotgun metabarcoding approach. The number of paired-end reads for this OTU accounted for 12.6% of the total number of ITS2 reads in this sample. Based on the analysis of the experimental and published ITS2 sequences of P. ginseng andP. quinquefolius , two stable SNPs (32 bp and 43 bp) were found and can be used to distinguish each other (Chen et al., 2013). A combination of C and T at positions 32 and 43 indicated there wasP. ginseng , while a combination of T and C indicated there wasP. quinquefolius . In order to confirm the existence of adulterant ingredients, P. quinquefolius , in HSZY192, we designed the taxon-specific primers of the ITS2 region for the two species. The trace data for PCR products of the mixed powder indicated that when theP. ginseng : P. quinquefolius content ratios were 1:9, 3:7, 5:5, and 7:3, different degrees of double peaks could be detected at the SNP positions (Figure 4 ). The results showed that the method can verify the existence of P. quinquefolius in the mixed powder of P. ginseng and P. quinquefolius . Subsequently, the PCR products ofpharmaceutical samples were sequenced and analyzed, and no double peaks were observed at the SNP positions. The combination of C and T was found at positions 32 and 43 from A03, HSZY152, and HSZY191, indicated that only the legal ingredient, P. ginseng , was detected in these samples. However, the combination of T and C was found at positions 32 and 43 from HSZY192, indicated that this sample does contain adulterant ingredient,P. quinquefolius , but not the labeled ingredients, P. ginseng (Supplementary Figure 3) . This result was consistent with that observed by the shotgun metabarcoding approach.