多重PCR同时鉴别5种药用海马
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摘要
海马是我国名贵动物药材之一,《中国药典》收载了5种海马的药用标准。随着海马资源储量的减少,市售海马基原增多,鉴别困难。该研究基于海马及其混伪品线粒体DNA序列差异设计了5对鉴别引物并构建同时鉴别三斑海马、克氏海马、管海马,刺海马和日本海马的多重PCR反应体系。PCR反应结束后进行凝胶电泳,管海马、日本海马、克氏海马、刺海马和三斑海马分别获得155, 222, 292, 352,458 bp的特异性鉴别条带,混伪品无条带。该研究建立的方法可以在单次PCR反应中准确鉴别所有药用海马的物种基原,为海马药材和饮片的质量控制提供了依据。
Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction(mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA,five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei(H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155,222,292,352,458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.
Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction(mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA,five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei(H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155,222,292,352,458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.
引文
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[2] 中国药典. 一部[S]. 2015: 293.
[3] 陈梦, 陈建真, 葛宇清, 等.海马化学成分及药理活性研究进展[J].中草药, 2017, 48(19): 4089.
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[10] 蒋超, 刘富艳, 金艳,等.基于形态和DNA序列分析的海马类药材商品的基原调查[J].中国中药杂志, 2018,43(23):4553.
[11] 吴平, 周开亚, 张朝晖,等.海马类药材的分子遗传标记鉴定研究[J].药学学报, 1998, 33(3):67.
[12] 张改霞, 刘金欣, 贾静, 等.基于COⅠ条形码的海马药材及其混伪品分子鉴定[J].中国药学杂志, 2015, 50(17):1469.
[13] 蒋超, 黄璐琦, 袁媛, 等. 《中国药典》动物药材基原物种中文名和拉丁学名引证规范[J]. 中国科学: 生命科学, 2018, 48(7): 772.
[14] Ward R D, Zemlak T S, Innes B H, et al. DNA barcoding Australia′s fish species[J]. Philos T R Soc B,2005,360: 1847.
[15] 陈志宣,龚国利,钱云开.阿胶DNA提取方法优化[J].中国实验方剂学杂志, 2014, 20(21):13.
[16] 蒋超, 黄璐琦, 袁媛, 等.使用碱裂解法快速提取药材DNA方法的研究[J].药物分析杂志, 2013, 33(7):1081.
[17] Jiang C, Luo Y, Yuan Y, et al. Conventional octaplex PCR for the simultaneous identification of eight mainstream closely related Dendrobium species[J]. Ind Crop Prod, 2018,112: 569.
[18] 刘富艳, 金艳, 袁媛, 等.多重位点特异性PCR鉴别海龙及其混伪品[J].中国实验方剂学杂志, 2018, 15(8):57.
[19] 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.
[20] 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.
[21] Lourie S A, Pollom R A, Foster S J. A global revision of the seahorses Hippocampus Rafinesque 1810 (Actinopterygii: Syngnathiformes): taxonomy and biogeography with recommendations for further research[J]. Zootaxa, 2016, 4146:1.
[22] Zhang Y H, Qin G, Wang X, et al. A new species of seahorse (Teleostei: Syngnathidae) from the South China Sea[J]. Zootaxa,2016, 4170: 384.
[2] 中国药典. 一部[S]. 2015: 293.
[3] 陈梦, 陈建真, 葛宇清, 等.海马化学成分及药理活性研究进展[J].中草药, 2017, 48(19): 4089.
[4] 姜素芬, 吉爱国, 梁浩, 等.我国海马的研究进展[J].中药材, 2007, 30(7): 884.
[5] 许东晖, 梅雪婷, 李秉记, 等.海马胶囊提高大鼠性功能的药理作用[J].中药材, 2003, 26(11):807.
[6] 陈建设,周东,孙自学,等.海马补肾壮阳丸治疗特发性弱精子症[J].中国实验方剂学杂志,2015,21(4):186.
[7] Vincent A C J. The international trade in seahorses[M]. Cambridge, UK: Traffic International,1996: 163.
[8] Chang C H, Jang-Liaw N H, Lin Y S, et al. Authenticating the use of dried seahorses in the traditional Chinese medicine market in Taiwan using molecular forensics[J]. J Food Drug Anal,2013, 21: 310.
[9] Sanders J G, Cribbs J E, Fienberg H G, et al. The tip of the tail: molecular identification of seahorses for sale in apothecary shops and curio stores in California[J]. Conserv Genet, 2008, 9(1): 65.
[10] 蒋超, 刘富艳, 金艳,等.基于形态和DNA序列分析的海马类药材商品的基原调查[J].中国中药杂志, 2018,43(23):4553.
[11] 吴平, 周开亚, 张朝晖,等.海马类药材的分子遗传标记鉴定研究[J].药学学报, 1998, 33(3):67.
[12] 张改霞, 刘金欣, 贾静, 等.基于COⅠ条形码的海马药材及其混伪品分子鉴定[J].中国药学杂志, 2015, 50(17):1469.
[13] 蒋超, 黄璐琦, 袁媛, 等. 《中国药典》动物药材基原物种中文名和拉丁学名引证规范[J]. 中国科学: 生命科学, 2018, 48(7): 772.
[14] Ward R D, Zemlak T S, Innes B H, et al. DNA barcoding Australia′s fish species[J]. Philos T R Soc B,2005,360: 1847.
[15] 陈志宣,龚国利,钱云开.阿胶DNA提取方法优化[J].中国实验方剂学杂志, 2014, 20(21):13.
[16] 蒋超, 黄璐琦, 袁媛, 等.使用碱裂解法快速提取药材DNA方法的研究[J].药物分析杂志, 2013, 33(7):1081.
[17] Jiang C, Luo Y, Yuan Y, et al. Conventional octaplex PCR for the simultaneous identification of eight mainstream closely related Dendrobium species[J]. Ind Crop Prod, 2018,112: 569.
[18] 刘富艳, 金艳, 袁媛, 等.多重位点特异性PCR鉴别海龙及其混伪品[J].中国实验方剂学杂志, 2018, 15(8):57.
[19] 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.
[20] 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.
[21] Lourie S A, Pollom R A, Foster S J. A global revision of the seahorses Hippocampus Rafinesque 1810 (Actinopterygii: Syngnathiformes): taxonomy and biogeography with recommendations for further research[J]. Zootaxa, 2016, 4146:1.
[22] Zhang Y H, Qin G, Wang X, et al. A new species of seahorse (Teleostei: Syngnathidae) from the South China Sea[J]. Zootaxa,2016, 4170: 384.