基于ITS序列位点特异性PCR鉴别桃仁与苦杏仁  

Identification of Persicae Semen and Armeniacae Semen Amarum by PCR Amplification of Specific Alleles Based on ITS Sequences

作　　者：高琳惠 尹艳 李佳[1] 马玥萌 李文斌[1] 李博文 张夏楠[1] Gao Linhui Yin Yan Li Jia Ma Yuemeng Li Wenbin Li Bowen Zhang Xia＇nan(School of Traditional Chinese Medcine, Capital Medical University, Beijing 100069, China School of Pharmaceutical Sciences, Guizhou University, Guiyang 550025, China)

机构地区：[1]首都医科大学中医药学院,北京100069 [2]贵州大学,贵阳550025

出　　处：《世界中医药》2017年第9期2190-2194,共5页World Chinese Medicine

基　　金：国家自然科学基金(81303166);国家中医药管理局中医药行业专项(201407003)

摘　　要：目的:对桃仁和苦杏仁进行分子鉴定研究,建立基于ITS序列位点特异性的PCR鉴定方法。方法:收集桃仁、苦杏仁样品,提取基因组总DNA,用ITS通用引物进行测序,通过Bio Edit软件进行序列比对,根据特异位点设计鉴别引物进行特异性PCR反应,并对PCR反应条件进行了优化。结果:基于ITS序列设计的特异性鉴别引物G4-7可以用于桃仁和苦杏仁的鉴别研究,在位点特异性PCR反应条件考察中,25μL反应体系DNA模板用量适宜范围为0.05~1 ng,退火温度在59℃时特异性最佳,实验建立的方法对不同DNA聚合酶和PCR仪均具有普适性。结论:该研究设计的位点特异性引物在一定条件下的PCR反应中,桃仁能够扩增出333 bp清晰条带,而苦杏仁没有该条带,从而实现了桃仁与苦杏仁的快速、准确鉴别。To identify Persicae semen amarum using DNA molecular identification,and establish classical PCR identification mesthod. The internal transcribed spacer（ ITS） of nuclear ribosomal DNA of Persicae Semen and Armeniacae semen amarum were amplified and bidirectionally sequenced. ITS sequences were analysed by Bio Edit. Specific identification primers were designed according to the ITS sequences of specific alleles,and PCR reaction system was optimized. Persicae Semen and Armeniacae Semen Amarum can be identificated by the specfic peimers G4-7 based on ITS sequences. In the condition’ s concentration is between0. 05-1 ng for a 25 μL PCR reaction,the best annealing temperature is 59 ℃. This method is well used for different DNA polymerases and different PCR instruments. The result showed that the 333 bp identification band can be amplified in Persicae semen,while it can not be amplified in Armeniacae semen amarum. It is confirmed that the PCR amplification system of specific alleles can be used to identify Persicae semen and Armeniacae semen amarum fastly and accurately.

关 键 词：桃仁 苦杏仁 ITS 位点特异性PCR 分子鉴定 

分 类 号：R284[医药卫生—中药学] R917[医药卫生—中医学]