蒙古黄芪、膜荚黄芪及混伪品种子的位点特异性PCR鉴别

Specific PCR for Identification of Astragalus membranaceus var.mongholicus Seeds,A.membranaceus Seeds,and Adulterants

目的:基于特异性聚合酶链式反应(PCR)建立一种可以准确、快速鉴别蒙古黄芪、膜荚黄芪种子的方法。方法:利用叶绿体基因组综合数据库(CGIR)及IdenDSS软件分别获取蒙古黄芪、膜荚黄芪的DNA特征片段,在此基础上筛选获得蒙古黄芪与膜荚黄芪的特异性单核苷酸多态性(SNP)位点,根据该位点设计蒙古黄芪特异性鉴别引物对MG-F/MG-R和膜荚黄芪特异性鉴别引物对MJ-F/MJ-R。分别建立蒙古黄芪和膜荚黄芪特异性PCR鉴别方法,优化PCR反应体系,并对该方法的专属性和适用性进行考察。结果:蒙古黄芪特异性PCR鉴别方法,采用引物对MG-F/MG-R、退火温度62℃、循环次数28次,经PCR扩增和凝胶电泳后蒙古黄芪在约220 bp处得到特异性条带,而膜荚黄芪和其他混伪品种子无条带;膜荚黄芪特异性PCR鉴别方法,采用引物对MJ-F/MJ-R、退火温度58℃、循环次数28次,经PCR扩增和凝胶电泳后膜荚黄芪扩增得到约150 bp的条带,而蒙古黄芪及混伪品无扩增产物。结论:该研究建立的特异性PCR鉴别方法,可以准确、快速对蒙古黄芪、膜荚黄芪进行种源鉴别,为黄芪种子的分类与准确鉴定提供了一定的科学依据。

Objective:To establish a method based on specific polymerase chain reaction(PCR)that can accurately and rapidly identify Astragalus membranaceus var.mongholicus(AMM)seeds and A.membranaceus(AM)seeds.Method:The Chloroplast Genome Information Resource(CGIR)and IdenDSS were used to obtain the characteristic DNA fragments of AMM and AM,and the specific single nucleotide polymorphism(SNP)sites of AMM and AM were screened out,on the basis of which the specific primers MG-F/MG-R of AMM and MJ-F/MJ-R of AM were designed.The specific PCR method for identifying AMM and AM was established and optimized,and the specificity and applicability of the method were investigated.Result:The specific PCR conditions for the identification of AMM were primers MG-F/MG-R,annealing at 62℃,and 28 cycles.After PCR amplification and gel electrophoresis,the specific band appeared at about 220 bp,with no band for the seeds of AM or adulterants.The specific PCR conditions for identifying the AM were primers MJ-F/MJ-R,annealing at 58℃,and 28 cycles.After PCR amplification and gel electrophoresis,the band appeared at about 150 bp,with no band of AMM or adulterants.Conclusion:The specific PCR method established in this study can accurately and quickly identify the seeds of AMM and AM,which provides a basis for the classification and accurate identification of Astragalus seeds and adulterants.