基于ITS序列PCR-RFLP鉴别木香、川木香和藏木香

Identification of Aucklandia Costus,Dolomiaea Souliei and Inula Racemosa by PCR-RFLP Based on ITS Sequence

目的利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术,建立快速鉴别木香、川木香和藏木香的方法。方法通过比对木香、川木香和藏木香3个物种ITS基因序列,筛选限制性内切酶并设计鉴别引物。优化PCR扩增和酶切反应条件,并进行方法学考察和统计学分析。结果退火温度为60℃、循环数为30时,能检出500~750 bp单一DNA条带;底物为10μL、酶切时间为15 min时,BsaHⅠ酶可将木香、川木香及藏木香在一定范围内切割成大小不一的DNA条带,方法学和统计学结果较好。结论建立的PCR-RFLP方法能够准确地鉴别木香、川木香及藏木香3种药材,避免此3类药材的混用,保证临床用药安全。

OBJECTIVE To establish a rapid method for distinguishing Aucklandia costus,Dolomiaea Souliei and Inula racemosa by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).METHODS The ITS gene sequences of Aucklandia costus,Dolomiaea souliei and Inula racemosa were compared,restriction enzymes were screened and identification primers were designed.The PCR amplification and digestion reaction conditions were optimized,and the methodological examinations and statistical analysis were carried out.RESULTS When the annealing temperature was 60℃and the cycle number was 30,a single DNA band of 500~750 bp could be detected.When the substrate was 10μL and the digestion time was 15 min,BsaHI enzyme could cut Aucklandia costus,Dolomiaea souliei and Inula racemosa into DNA bands of different sizes in a certain range.The results of methodology and statistics were good.CONCLUSION The PCR-RFLP method established in this study can accurately identify the three medicinal materials of Aucklandia costus,Dolomiaea souliei and Inula racemosa,avoid the mixed use of these three medicinal materials,and ensure the safety of the clinical medication.