多重位点特异性PCR鉴别海龙及其混伪品
|
摘要
目的:建立一种高效、准确的中药材海龙及其常见混伪品的特异性聚合酶链式反应(polymerase chain reaction,PCR)鉴别方法。方法:通过比较海龙及其混伪品的细胞色素C氧化酶亚基Ⅰ基因(cytochrome C oxidase subunitⅠ,COⅠ)基因序列差异,根据变异位点设计刁海龙、尖海龙及拟海龙的特异性鉴别引物,优化反应体系,并对此方法进行耐受性和适用性的考察和验证。在此基础上,将3对特异性引物组合,构建多重PCR体系。结果:在多重PCR体系中,刁海龙能扩增出485 bp片段,尖海龙可扩增出120 bp片段,拟海龙可以扩增出240 bp片段,其他混伪品均无条带。所设计的特异性鉴别引物具有高度的特异性。结论:该文所建立的位点特异性PCR鉴别方法可实现海龙的准确鉴别。
Objective: To establish an effective and accurate specific polymerase chain reaction(PCR)identification method for herb Syngnathus and its common adulterates. Method: Based on the difference in cytochrome C oxidase subunit Ⅰ(CO Ⅰ) gene DNA sequences among Solenognathus hardwickii,Syngnathoides biaculeatus,S. acus and adulterants,the specific primers were designed; the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility. The three pairs of specific primers were combined to build multiplex PCR systems. Result: Through the established multiplex PCR reaction system,485,240,120 bp fragments were amplified from DNA templates of S. shardwickii,S. biaculeatus and S. acus, respectively. All the adulterants had no bands. Conclusion: The designed identification primers were highly specific,and the PCR amplification of specific alleles established in this paper can be used to accurately identify the Syngnathus.
Objective: To establish an effective and accurate specific polymerase chain reaction(PCR)identification method for herb Syngnathus and its common adulterates. Method: Based on the difference in cytochrome C oxidase subunit Ⅰ(CO Ⅰ) gene DNA sequences among Solenognathus hardwickii,Syngnathoides biaculeatus,S. acus and adulterants,the specific primers were designed; the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility. The three pairs of specific primers were combined to build multiplex PCR systems. Result: Through the established multiplex PCR reaction system,485,240,120 bp fragments were amplified from DNA templates of S. shardwickii,S. biaculeatus and S. acus, respectively. All the adulterants had no bands. Conclusion: The designed identification primers were highly specific,and the PCR amplification of specific alleles established in this paper can be used to accurately identify the Syngnathus.
引文
[1]张朝晖,徐国钧,徐珞珊,等.海龙类药材性状与商品鉴定[J].中药材,1997,20(2):63-66.
[2]税丕先.海龙及其混淆品掺假品的鉴别[J].中国药业,2004,13(7):59-60.
[3]刘富艳,秦雯,袁媛,等.基于形态和DNA序列分析的海龙类药材商品的基原调查[J].世界中医药,2018,13(2):18-23.
[4]魏艺聪,蒋超,袁媛,等.基于COⅠ与SRY序列建立梅花鹿、马鹿及其杂交鹿茸的分子鉴别方法[J].中国中药杂志,2017,42(23):4588-4592.
[5]尹艳,刘逊,王兵,等.中药穿山甲的DNA分子鉴定研究[J].中国中药杂志,2017,42(11):2078-2084.
[6]蒋超,屠李婵,袁媛,等.金银花配方颗粒的位点特异性PCR鉴别研究[J].中国中药杂志,2017,42(13):2484-2491.
[7]蒋超,罗宇琴,袁媛,等.多重位点特异性PCR鉴别人参、三七、西洋参掺杂[J].中国中药杂志,2017,42(7):1319-1323.
[8]蒋超,赵群,金艳,等.快速PCR技术鉴别中药材蛤蚧的方法研究[J].中国现代中药,2017,19(1):21-25.
[9]王凤云,蒋颖诗,赖小平.基于ND2基因序列的燕窝DNA条形码鉴别[J].中国实验方剂学杂志,2015,21(13):36-40.
[10]吴艳,刘佳,王梦月,等.海龙及其常见伪品的RAPD鉴别[J].中国中药杂志,2009,34(14):1758-1760.
[11]GAO L,YIN Y,LI J,et al.Identification of traditional Chinese medicinal pipefish and exclusion of common adulterants by multiplex PCR based on 12 S sequences of specific alleles[J].Mitochondrial DNA A,2017,doi:10.1080/24701394.2016.1278538.
[12]ZHANG Y H,QIN G,ZHANG H X,et al.DNA barcoding reflects the diversity and variety of brooding traits of fish species in the family syngnathidae along China's coast[J].Fish Res,2017,doi.org/10.1016/j.fishres.2016.09.015.
[13]陈明洁,方倜,柯涛,等.多重PCR——一种高效快速的分子生物学技术[J].武汉理工大学学报,2005,27(10):37-40.
[14]黄璐琦,袁媛.中药分子鉴定操作指南[M].上海:上海科学技术出版社,2014:49.
[2]税丕先.海龙及其混淆品掺假品的鉴别[J].中国药业,2004,13(7):59-60.
[3]刘富艳,秦雯,袁媛,等.基于形态和DNA序列分析的海龙类药材商品的基原调查[J].世界中医药,2018,13(2):18-23.
[4]魏艺聪,蒋超,袁媛,等.基于COⅠ与SRY序列建立梅花鹿、马鹿及其杂交鹿茸的分子鉴别方法[J].中国中药杂志,2017,42(23):4588-4592.
[5]尹艳,刘逊,王兵,等.中药穿山甲的DNA分子鉴定研究[J].中国中药杂志,2017,42(11):2078-2084.
[6]蒋超,屠李婵,袁媛,等.金银花配方颗粒的位点特异性PCR鉴别研究[J].中国中药杂志,2017,42(13):2484-2491.
[7]蒋超,罗宇琴,袁媛,等.多重位点特异性PCR鉴别人参、三七、西洋参掺杂[J].中国中药杂志,2017,42(7):1319-1323.
[8]蒋超,赵群,金艳,等.快速PCR技术鉴别中药材蛤蚧的方法研究[J].中国现代中药,2017,19(1):21-25.
[9]王凤云,蒋颖诗,赖小平.基于ND2基因序列的燕窝DNA条形码鉴别[J].中国实验方剂学杂志,2015,21(13):36-40.
[10]吴艳,刘佳,王梦月,等.海龙及其常见伪品的RAPD鉴别[J].中国中药杂志,2009,34(14):1758-1760.
[11]GAO L,YIN Y,LI J,et al.Identification of traditional Chinese medicinal pipefish and exclusion of common adulterants by multiplex PCR based on 12 S sequences of specific alleles[J].Mitochondrial DNA A,2017,doi:10.1080/24701394.2016.1278538.
[12]ZHANG Y H,QIN G,ZHANG H X,et al.DNA barcoding reflects the diversity and variety of brooding traits of fish species in the family syngnathidae along China's coast[J].Fish Res,2017,doi.org/10.1016/j.fishres.2016.09.015.
[13]陈明洁,方倜,柯涛,等.多重PCR——一种高效快速的分子生物学技术[J].武汉理工大学学报,2005,27(10):37-40.
[14]黄璐琦,袁媛.中药分子鉴定操作指南[M].上海:上海科学技术出版社,2014:49.