Identification of plant materials containing ephedrine alkaloids based on DNA barcoding and TaqMan real-time PCR assay

An intentional or inadvertent mixing of plant materials containing ephedrine alkaloids, especially Ephedra , is illegal. In order to better detect plant materials containing ephedrine alkaloids in export and smuggling, DNA barcoding combined with a TaqMan real-time PCR-based assay were used in this study. We collected 201 samples from 18 species belonging to four genera distributed in three families to amplify two barcoding markers, internal transcribed spacer 2 (ITS2) and psb A -trn H. 175 ITS2 sequences and 136 psb A- trn H sequences were obtained. Alignments and the neighbor-joining tree indicated that ITS2 showed a better discrimination of species than psb A -trn H. In addition, based on the sequence comparison of the ITS2 region from 18 species, three sets of primer/probes were designed using the real-time PCR assay platform. A sensitivity test showed the above primer/probe sets were specific and sensitive (as little as 0.0050 ng/μL target DNA) to identify species containing ephedrine alkaloids. Hence, the combination of novel DNA barcoding and the TaqMan real-time PCR-based technique are promising tools for the identification of plant materials containing ephedrine alkaloids. It is beneficial to standardize market circulation and detection of plant materials containing ephedrine alkaloids.