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.