基于ITS序列PCR-RFLP鉴别大蓟药材及饮片中掺伪飞廉、魁蓟的方法研究

Identification of Cirsium japonicum,Carduus nutans and Cirsium leo by specific PCR-RFLP

[目的]利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP),建立快速鉴别大蓟混伪品飞廉和魁蓟的方法。[方法]通过比对ITS基因序列,分别筛选飞廉、魁蓟的限制性内切酶位点并设计鉴别引物。考察PCR反应的退火温度、循环数及不同酶的适用性,对酶切反应时间和酶切底物量进行优化。同时对该方法适应性及掺伪比例的专属性、稳定性进行考察。[结果]当退火温度为58~60℃、循环数为30个时,样品均能扩增出一条379 bp的DNA条带。底物为8μL、酶切温度为37℃、酶切反应120 min时,ZraI酶将飞廉切割成120 bp和259 bp两条DNA条带;DNA底物为8μL、酶切温度为37℃、酶切反应60 min时,ApaLI酶将魁蓟切割成126 bp和253 bp两条DNA条带。[结论]试验建立的PCR-RFLP方法能够准确地鉴别大蓟混伪品飞廉、魁蓟,避免此类药材的混用,以保证临床用药安全。

[Objective]To establish a rapid method to identify the adulterants of Cirsium japonicum:Carduus nutans and Cirsium leo by polymerase chain reaction restriction fragment length polymorphism(PCR-RFLP).[Methods]By comparing the ITS gene sequences,the restriction endonucleases of Carduus nutans and Cirsium leo were screened and the primers were designed.The annealing temperature,cycle number and applicability of different enzymes of PCR reaction were investigated,and the digestion time and substrate amount were optimized.At the same time,the adaptability,specificity and stability of the method were investigated.[Results]When the annealing temperature is 58-60℃and the number of cycles is 30,a 379 bp DNA band can be amplified from the sample.Substrate is 8μL.When the digestion temperature was 37℃and the digestion reaction was 120 min,ZraI enzyme cut two DNA bands of 120 bp and 259 bp from Carduus nutans;DNA substrate is 8μL.When the digestion temperature was 37℃and the digestion reaction was 60 min,ApaLI enzyme cut two DNA bands of 126 bp and 253 bp from Cirsium leo.[Conclusion]The PCR-RFLP method established in this study can accurately identify the adulterants of Cirsium japonicum:Carduus nutans and Cirsium leo,avoid the mixing of these herbs,and ensure the safety of clinical medication.