基于MLPA结合熔解曲线法鉴别山药及其常见混伪品
Identification of Dioscoreae Rhizoma and Common Adulterants Based on MLPA Combined with Melting Curve Method
李小芳 1王文斌 1莫静 2程华春 1谢云 3汪波4
作者信息
- 1. 湖北省药品监督检验研究院,湖北 武汉 430075;湖北中医药大学,湖北 武汉 430065
- 2. 湖北省药品监督检验研究院,湖北 武汉 430075;湖北大学,湖北 武汉 430062
- 3. 湖北中医药大学,湖北 武汉 430065
- 4. 湖北省药品监督检验研究院,湖北 武汉 430075;国家药品监督管理局中药质量控制重点实验室,湖北 武汉 430075
- 折叠
摘要
目的:利用MLPA技术结合熔解曲线法建立一种能鉴别山药及其常见混伪品的新方法.方法:以山药、参薯、木薯、番薯为研究对象,针对四个物种的matK序列分别设计MLPA特异性探针,通过DNA变性、杂交、连接、扩增及熔解反应等步骤,建立MLPA-熔解曲线鉴别体系,利用各样品熔解曲线中特异性峰(Tm值)的差异实现不同物种的鉴定.结果:27份样品中,山药Tm值为78.3 ℃,参薯Tm值为79.9℃,木薯Tm值为81.2℃,番薯Tm值为82.9℃,且相互之间没有交叉干扰.该法对山药、参薯、木薯、番薯各自单独的最低检测限为0.1 ng/μL;对相互混淆掺伪的最低有效检出率为5％.27份样品的检测结果均与测序结果一致.结论:MLPA技术结合熔解曲线法具有特异性强、灵敏度高、操作简单等优点,具有进一步推广应用的潜力.
Abstract
Objective:To establish an identification method for Dioscoreae Rhizoma and its common adulterants by using MLPA technique and melting curve.Methods:With Dioscoreae Rhizoma and the tubers of Dioscorea alata,Manihot esculenta,Ipomoea batatas as the research object,MLPA-specific probes were designed for matK sequences of four species.MLPA-fusion curve identification sys-tem was established by DNA denaturation,hybridization,ligation,amplification and fusion reaction,respectively.The identification of dif-ferent species was achieved by using the difference of specific peaks(Tm values)in the melting curves of each sample.Results:Among the 27 samples,the Tm value of Dioscoreae Rhizoma was 78.3 ℃,the Tm value of Dioscorea alata tubers was 79.97 ℃,the Tm value of Manihot esculenta tubers was 81.2 ℃ and the Tm value of Ipomoea batatas tubers was 82.9 ℃,and there was no cross interference between each other.The lowest detection limit of Dioscoreae Rhizoma,and the tubers of Dioscorea alata,Manihot esculenta,Ipomoea batatas was 0.1 ng/μL.The minimum effective detection rate for mutual confusion was 5％.The results of 27 samples were consistent with the sequencing results.Conclusion:MLPA technology combined with melting curve method has the advantages of strong specificity,high sensitivity and simple operation,so it has the potential to be further popularized and applied.
关键词
山药/多重连接探针扩增技术/熔解曲线/matK/鉴别Key words
Dioscoreae Rhizoma/Multiplex ligation-dependent probe amplification(MLPA)/Melting curve/matK/Identify引用本文复制引用
基金项目
湖北省青年拔尖人才培养计划(2021)()
&&(2020ACA007)
中国食品药品检定研究院项目(TCM2021-YJ11)
出版年
2024
国家科技期刊平台
NSTL
万方数据