基于DSS标记特异性PCR鉴别冷背药材木槿皮基原植物及其混伪品

Specific PCR Identification of Hibisci Cortex and Its Adulterants Based on DNA Signature Sequence Tags

目的:冷背药材作为宝贵的药材资源,但因其本身的特殊性和复杂性,导致其鉴别成为中药鉴定中的难题,因此亟需建立冷背药材的鉴别方法。该研究利用DNA特征序列(DSS)标记,建立一种冷背药材木槿皮基原植物木槿及其混伪品的特异性聚合酶链式反应(PCR)鉴别方法。方法:基于叶绿体基因组序列分析获得木槿皮的候选DSS标记,使用DNA测序手段对DSS标记进行验证,基于得到的可靠DSS标记设计木槿皮基原植物特异性鉴别引物,对PCR反应条件进行优化,并进行耐受性和适用性的考察。结果:将扩增产物的测序结果与DSS比对后得到了1个可用于鉴定木槿的DSS标记,揭示了木槿正混伪品的区别特征,根据DSS标记设计出一对木槿正品特异性鉴别引物,使用该引物进行PCR扩增和凝胶电泳后木槿均出现约270 bp的单一明亮条带,而其4种混伪品均无此条带。结论:该研究得到的1个DSS标记可用于木槿的真伪鉴定,基于此DSS标记设计的特异性鉴别引物可准确、简便的鉴别木槿皮的基原植物及其混伪品,为木槿正伪品的分子鉴定提供了新的方法与思路。

Objective:Uncommon medicinal herbs are valuable medicinal resources,but their identification is a difficult problem in Chinese medicine due to their particularity and complexity.It is,therefore,urgent to establish a method for the identification of uncommon medicinal herbs.In this study,DNA signature sequence(DSS)tags were used to establish a specific polymerase chain reaction(PCR)identification method for Hibisci Cortex,the origin plant of Hibisci Cortex,and its adulterants.Method:The candidate DSS tags were obtained from the chloroplast genome sequence analysis,and the DSS tags were verified by DNA sequencing.The specific identification primers for H.syriacus were designed based on the obtained reliable DSS tags.The PCR reaction conditions were optimized,and the tolerance and feasibility were investigated.Result:A DSS tag for identification of H.syriacus was obtained from the comparison of sequencing results of the amplified products with DSS,which revealed the distinguishing characteristics of Hibisci Cortex and its adulterants.A pair of specific primers for H.syriacus was designed according to the DSS tag.After PCR amplification and gel electrophoresis with the primers,a single bright band of about 270 bp was observed from H.syriacus,which did not appear in the four adulterants.Conclusion:A DSS tag obtained in this study can be used to identify H.syriacus.The specific primers designed based on this DSS tag can accurately and simply identify the original plant of Hibisci Cortex and its adulterants,which provides a new method and idea for the molecular identification of genuine and counterfeit products of Hibisci Cortex.