多重连接探针扩增-高分辨熔解法鉴别鸡血藤及其常见混伪品  

Identification of Spatholobus suberectus and Its Common Adulterants by MLPA-HRM Assay

作　　者：王咏懿 莫静 王文斌 程华春 李小芳 汪波 WANG Yongyi;MO Jing;WANG Wenbin;CHENG Huachun;LI Xiaofang;WANG Bo(School of Pharmacy,China Pharmaceutical University,Nanjing 211112,China;Hubei Institute for Drug Control,Wuhan 430075,China;School of Life Science,Hubei University,Wuhan 430062,China;School of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China;NMPA Key Laboratory of Quality Control of Chinese Medicine,Wuhan 430075,China)

机构地区：[1]中国药科大学药学院,南京211112 [2]湖北省药品监督检验研究院,武汉430075 [3]湖北大学生命科学学院,武汉430062 [4]湖北中医药大学药学院,武汉430065 [5]国家药品监督管理局中药质量控制重点实验室,武汉430075

出　　处：《医药导报》2023年第3期334-339,共6页Herald of Medicine

基　　金：湖北省青年拔尖人才培养计划;湖北中药质控标准物质研制及技术平台的建立(2020ACA007);中国食品药品检定研究院项目(TCM2021-YJ11)。

摘　　要：目的采用分子生物学技术鉴别鸡血藤及其常见3种混伪品。方法根据4个物种核基因组内转录间隔区2(ITS2)序列差异设计特异性探针,结合运用多重连接探针扩增(MLPA)技术与高分辨熔解(HRM)曲线技术,根据特异性探针熔解温度(T m)差异鉴别鸡血藤及其常见混伪品大血藤、白花油麻藤、香花鸡血藤。结果4个物种特异性探针T m值分别为:鸡血藤(87.0±0.2)℃、大血藤(82.8±0.2)℃、白花油麻藤(85.4±0.2)℃、香花鸡血藤(80.1±0.2)℃。探针特异性考察中,交叉反应与空白组均未产生熔解峰,表明探针具有良好特异性。构建的方法可用于1.0 ng模板DNA的检测,并可有效检出鸡血藤中掺混5%大血藤、白花油麻藤或香花鸡血藤。结论基于MLPA-HRM技术建立的鸡血藤及其混伪品鉴别方法灵敏度高,特异性强,稳定性好,可有效解决市场上鸡血藤药材植物来源复杂、混伪品难以鉴别的问题。Objective To identify Spatholobus suberectus and three kinds of its common adulterations with molecular biology techniques.Methods Specific probes were designed according to different ITS2 sequences of four species.Multiple linked probe amplification(MLPA)technology and high resolution melting(HRM)curve technology were used to identify Spatholobus suberectus and its common adulterants,Sargentodoxa cuneate,Mucuna birdwoodiana,or Callerya dielsiana,according to the difference of specific probe melting temperature(T m).Results The specific probe T m values of the four species were(87.0±0.2)℃for Spatholobus suberectus,(82.8±0.2)℃for Sargentodoxa cuneate,(85.4±0.2)℃for Mucuna birdwoodiana,and(80.1±0.2)℃for Callerya dielsiana,respectively.In probe-specific investigations,the cross-reactive group and the blank group did not produce melting peaks,indicating that the probes have good specificity.The established method can be used for the detection of 1.0 ng of template DNA,and can effectively detect 5%of Sargentodoxa cuneate,Mucuna birdwoodiana and Callerya dielsiana mixed in Spatholobus suberectus.Conclusion The established method for identifying Spatholobus suberectus and its adulterants based on MLPA-HRM technology has high sensitivity,strong specificity and good stability,which can effectively solve the problems of complex plant sources and difficult identification of adulterants in the market.

关 键 词：鸡血藤 混伪品 多重连接探针扩增技术 高分辨熔解曲线 鉴别 

分 类 号：R286[医药卫生—中药学] R927.1[医药卫生—中医学]