Discriminating between Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations by nested PCR and DNA sequencing methods

The widely used Achyranthis Bidentatae Radix (Niu Xi) in Chinese medicine preparation is easily misused and confused with Cyathulae Radix (Chuan Niu Xi). Taiwan authority has ruled that Achyranthis Bidentatae Radix and Cyathulae Radix should be individually entered in the given preparations. In this study, with the internal transcribed spacer (ITS) as a DNA marker, nested PCR and DNA sequencing were used to distinguish the two herbs in preparations. Authentic Achyranthis Bidentatae Radix and Cyathulae Radix were collected from the original country, mainland China, and twenty two samples of Chinese medicine preparations labeled with Niu Xi as the ingredient were purchased locally. The total DNA of all samples were extracted by organic solution and purified using a kit. For a standard, ITS regions of authentic Achyranthis Bidentatae Radix and Cyathulae Radix were amplified with PCR and analyzed by an auto-sequencer. The ITS regions of preparation samples of DNA were amplified by nested PCR and the data of sequences received from an auto-sequencer. We compared the ITS sequences of preparation samples with the sequences of a standard and Genbank database to check which Achyranthis Bidentatae Radix or Cyathulae Radix is in the preparation samples. The results showed that only one sample contained Achyranthis Bidentatae Radix and the others contained Cyathulae Radix. The Cyathulae Radix components in four preparation samples do not fit into Taiwan authority rules. And our method can be applied for the differentiation of Achyranthis Bidentatae Radix and Cyathulae Radix in Chinese medicine preparations.