基于ITS序列位点特异性PCR鉴别桃仁与苦杏仁
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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.
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.
引文
[1]国家药典委员会.中华人民共和国药典(一部)[M].北京:中国医药科技出版社,2015:201-202,277-278.
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[14]Li DZ,Gao LM,Li HT,et al.Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants[J].Proc Natl Acad Sci U S A,2011,108(49):19641-19646.
[2]杜虹韦,张爱华,赵欣蕾.苦杏仁毒性及其解毒方法研究进展[J].黑龙江中医药,2013,43(4):58-59.
[3]Chen S,Yao H,Han J,et al.Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plant species[J].PLo S One,2010,5(1):e8613.
[4]黄璐琦.中药分子鉴定操作指南[M].上海:上海科学技术出版社,2014.
[5]蒋超,侯静怡,黄璐琦,等.快速PCR方法在金银花真伪鉴别中的应用[J].中国中药杂志,2014,39(19):3668-3672.
[6]蒋超.中药现场、快速鉴别方法的建立—以金银花为例[D].北京:中国中医科学院,2013.
[7]李振华,龙平,邹德志,等.基于ITS序列位点特异性PCR的肉苁蓉与其混伪品的鉴别研究[J].中国中药杂志,2014,39(19):3684-3688.
[8]陈康,蒋超,袁媛,等.快速PCR方法在蛇类药材真伪鉴别中的应用[J].中国中药杂志,2014,39(19):3673-3677.
[9]赵丹,周涛,江维克,等.太子参药材的快速分子鉴定[J].中国中药杂志,2014,39(19):3689-3694.
[10]韦健红,吴文如,喻良文,等.DNA条形码技术在生物分类中的应用[J].广东药学院学报,2010,26(4):430-433.
[11]裴男才,陈步峰.生物DNA条形码:十年发展历程、研究尺度和功能[J].生物多样性,2013,21(5):616-627.
[12]韩建萍,宋经元,姚辉,等.中药材DNA条形码鉴定的基因序列比较[J].中国中药杂志,2012,37(8):1056-1061.
[13]程龙,葛斐林,刘泽邦,等.中药资源DNA条形码鉴定方法研究进展[C].北京:第四届中国中药商品学术大会暨中药鉴定学科教学改革与教材建设研讨会论文集,2015:398-404.
[14]Li DZ,Gao LM,Li HT,et al.Comparative analysis of a large dataset indicates that internal transcribed spacer(ITS)should be incorporated into the core barcode for seed plants[J].Proc Natl Acad Sci U S A,2011,108(49):19641-19646.