Identification of Levisticum officinale Adulterated in Angelica sinensis by PCR-RFLP / 中国实验方剂学杂志

Objective:To establish a rapid method to identify <italic>Levisticum officinale </italic>adulterated in<italic> Angelica sinensis</italic> by polymerase chain reaction -restriction fragment length polymophism（PCR-RFLP）. Method:By comparing sequences restriction sites in ribosomal DNA Internal Transcribed Spacer（ITS） of <italic>A. sinensis</italic> and <italic>L. officinale</italic>，the specific restriction site Fnu4HI of <italic>L. officinale</italic> was selected，and the primers for PCR-RFLP reaction were designed. Different <italic>A. sinensis</italic> and <italic>L. officinale</italic> were amplified by PCR. The conditions affecting the PCR-RFLP reaction，such as annealing temperature，primer concentration，cycle number and enzyme digestion reaction time，were optimized，and the accuracy of the method was investigated. The established PCR-RFLP identification method was used to investigate the applicability of <italic>L. officinale </italic>adulterated<italic> in A. sinensis</italic> with different aduleration ratios and different origins. Result:A PCR-RFLP method for identifying <italic>A. sinensis</italic> mixed with <italic>L. officinale</italic> was established. When the annealing temperature was 62 ℃ and the number of cycles was 30，when the <italic>L. officinale </italic>adulterated in<italic> A. sinensis</italic> could be digested by Fnu4H I restriction endonuclease after amplification with specific primers，and the two single DNA bands were detected between 100-500 bp，the <italic>A. sinensis</italic> were all negative. The minimum detection limit of this method for adulterated <italic>L. officinale</italic> in <italic>A. sinensis</italic> was 3%，which could be used for the detection of adulterated <italic>L. officinale</italic> in <italic>A. sinensis</italic>. Conclusion:The established PCR-RFLP identification method is sensitive and accurate in detecting whether there is <italic>L. officinale</italic> in <italic>A. sinensis</italic>，and it provides inspection reference and basis for the quality control of <italic>A. sinensis</italic>，with great significance to ensure the safety of its clinical medication.

Identification of Dendrobium huoshanense, Dendrobium officinale and Dendrobium devonianum by multiplex allele-specific polymerase chain reaction / 药学学报

This study was designed to establish a multiplex allele-specific polymerase chain reaction method for simultaneous identification of Dendrobium huoshanense, D. officinale and D. devonianum, which may resolve identification problems of caulis dendrobii. Internal transcribed spacer sequences and trnL-trnF sequences of the Dendrobium species were aligned by BioEdit software, then specific SNPs of the three species were analyzed for designing allele-specific primers and the multiplex allele-specific PCR reaction system was established. The different origin of Dendrobium huoshanense, D. officinale and D. devonianum was amplified and identified by the sizes of respective band. The results showed that 584 bp, 397 bp and 211 bp bands could be amplified by D. devonianum, Dendrobium officinale and Dendrobium huoshanense respectively, when the annealing temperature was 61 ℃ and the number of cycles was 35. The limit of detection (LOD) of D. devonianum and D. huoshanense were both 1.2 ng, while D. officinale was low than 0.24 ng. The detection limit of adulterates in D. devonianum, D. devonianum and D. huoshanense mixture sample was 1%, 1% and 5% respectively. This result suggests that the method of multiplex allele-specific PCR is useful to identify D. huoshanense, D. officinale and D. devonianum is accurate and specific.