目的: 通过聚合酶链反应-限制性片段长度多态性方法(PCR-RFLP法)建立一种能快速鉴别麦冬药材及饮片中掺混山麦冬的方法。方法: 对麦冬和山麦冬的核糖体DNA内转录间隔区(ITS)序列酶切位点进行比对,选择山麦冬的特异性酶切位点PmlI,设计PCR-RFLP反应引物。以退火温度58 ℃、循环数为30,LR-F和LR-R鉴别引物(10 μmol·L-1)各0.5 μL,PmlI限制性内切酶在37 ℃酶切2 h的反应条件为PCR-RFLP法的参数,并进行方法学考察和统计分析。结果: 在退火温度58 ℃、循环数为30时,山麦冬或掺有山麦冬的麦冬样品经过特异性引物扩增后,能被PmlI限制性内切酶酶切,在100~250 bp之间检出2条单一DNA条带,麦冬样品则无此条带。该方法稳定性好,专属性强,能对麦冬与山麦冬饮片及掺混样品进行鉴别,并对麦冬中掺入山麦冬的检出限为5%。结论: 建立的PCR-RFLP鉴别方法稳定性好,灵敏度高,实现了麦冬与山麦冬饮片及麦冬中掺混山麦冬的快速检测,可用于麦冬与山麦冬药材真伪鉴别的补充检验方法。
Objective: To establish a method for rapid identification of Liriopes Radix adulterated in Ophiopogon Radix medicinal materials and decoction pieces by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Methods: By comparison ITS sequences restriction site of Ophiopogonis Radix and Liriopes Radix, the specific restriction site of Liriopes Radix were selected, and the primers for PCR-RFLP reaction were designed. The different Ophiopogonis Radix and Liriopes Radix were amplified by PCR, PCR-RFLP reaction system annealing temperature, primer concentration, cycle number, enzyme digestion reaction time were and restriction endonuclease type optimized, and accuracy of the method were investigated. In addition, the PCR-RFLP method which had established was used to investigate the applicability of Ophiopogonis Radix, Liriopes Radix and adulterated sample from different sources. Results: A PCR-RFLP method for identifying Ophiopogonis Radix mixed with Liriopes Radix was established with the annealing temperature at 58 ℃ and the number of cycles of 30. Liriopes Radix and Liriopes Radix adulterated in Ophiopogonis Radix could be digested by PmlI restriction endonuclease after amplification with specific primers, the two single DNA bands were detected between 100-250 bp, which were not found in Ophiopogonis Radix. This method could be used to identify Ophiopogonis Radix, Liriopes Radix and the adulterated sample, and the minimum detection limit of this method for Liriopes Radix adulterated in Ophiopogonis Radix was 5%. Conclusion: The established PCR-RFLP method is specific and good stable, can be applied to the qualitatively identify Ophiopogonis Radix, Liriopes Radix and its adulterated sample, which can be used to establish supplem entary method.
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