目的: 建立中药材三斑海马DNA指纹鉴定方法,研制三斑海马DNA检测试剂盒。方法: 利用生物信息学对海马种属的Cytb基因进行分析,以该基因作为靶基因,设计特异性引物,研制三斑海马DNA提取及PCR快速检测试剂,应用分子克隆及测序技术,克隆三斑海马阳性对照药材,并对快速检测试剂盒的特异性、稳定性、重复性及灵敏度进行考察,对市售三斑海马样品进行真伪鉴定。结果: 基于Cytb序列可对三斑海马及其混伪品进行区分。自主研发的三斑海马快速检测试剂提取的海马DNA纯度和浓度均可达到PCR的要求,在退火温度为61.5 ℃时引物特异性最强;克隆测序后三斑海马DNA序列与海马mtDNA Cytb特异指纹区段序列一致;自主研制的试剂重现性、稳定性良好,检测浓度最低限为10-3ng·μL-1;对17个市售样品进行检测,合格率为88.2%。结论: 建立了一种特异、准确的检测中药材三斑海马DNA指纹鉴定方法;研制的三斑海马DNA检测试剂盒操作简便,灵敏度高,检测结果稳定。
[1] 中华人民共和国药典 2015 年版. 四部[S]. 2015:182
ChP 2015. Vol Ⅳ[S].2015:182
[2] 于曼曼. 三斑海马长链碱的制备及体外抗肿瘤作用的研究[D].海口:海南大学, 2018
YU MM. Study on the Preparation of Hippocampus trimaculatus Long Chain Alkaloids and its Antitumor Effect In Vitro[D].Haikou:Hainan University, 2018
[3] 陈莉萍. 三斑海马中抗炎、抗肿瘤、抗氧化活性成分的分离及生理活性评价研究[D].海口:海南大学, 2016
CHEN LP. Isolation and Physiological Activity Evaluation of Anti-Inflammatory, Anti-Tumor and Antioxidant Active Components in Hippocampus trimaculatus[D].Haikou:Hainan University, 2016
[4] 唐海婷,张才,张永平,等. 雌、雄海马抗疲劳和改善记忆障碍比较[J].广东海洋大学学报, 2019, 39(3):103
TANG HT, ZHANG C, ZHANG YP, et al. Comparison of female and male hippocampus in anti fatigue and improving memory impairment[J].J Guangdong Ocean Univ, 2019, 39(3):103
[5] 戴广谱. 环境因素对大海马的繁殖行为及其幼体生长的影响[D].宁波:宁波大学, 2012.
DAI GP. Effects of Environmental Factors on Reproductive Behavior and Larval Growth of Hippocampus kelloggi[D].Ningbo:Ningbo University, 2012
[6] 徐永健,陆慧贤,卢光明. 三斑海马的人工生态养殖[J].渔业科学进展, 2011, 32(5):38
XU YJ, LU HX, LU GM. Artificial ecological culture of Hippocampus trimaculatus[J].Progr Fishery Sci, 2011, 32(5):38
[7] 骆大鹏,刘庆明,邱名毅, 等. 三斑海马的驯化与苗种培育研究[J].安徽农业科学, 2016, 44(31):147
LUO DP, LIU QM, QIU MY, et al. Domestication and seedling cultivation of Hippocampus trimaculatus[J].J Anhui Agric Sci, 2016, 44(31):147
[8] 周胜杰,胡静,杨蕊,等. 三斑海马早期发育观察[J].南方农业学报, 2021,52(8):2302
ZHOU SJ, HU J, YANG R, et al. Observation on the early development of Hippocampus trimaculatus[J].J Southern Agric, 2021,52(8):2302
[9] 陈梦,朱玲燕,黄真,等. 三斑海马及其混伪品的DNA条形码分子鉴定研究[J].中草药, 2019, 50(22):5554
CHEN M, ZHU LY, HUANG Z, et al. Study on DNA bar code molecular identification of Hippocampus trimaculatus and its adulterants[J].Chin Tradit Herb Drugs, 2019, 50(22):5554
[10] 温珑莲,李军德,万德光,等. 海马市场调查与基原动物鉴定研究[J].中国中药杂志, 2013, 38(7):969
WEN LL, LI JD, WAN DG, et al. Market survey of hippocampus and identification of primitive animals[J].China J Chin Mater Med, 2013, 38(7):969
[11] 周胜杰,胡静,杨其彬,等. 三斑海马骨骼染色方法改进及骨骼结构特点研究[J].南方农业学报, 2021, 52(5):1387
ZHOU SJ, HU J, YANG QB, et al. Study on the improvement of bone staining method and the characteristics of bone structure in Hippocampus trimaculatus[J].J Southern Agric, 2021, 52(5):1387
[12] 何旭辉,张成中,黄宝康,等. 以微观性状鉴别为基础的海马分类研究[J].中国海洋药物, 2021, 40(4):18.
HE XH, ZHANG CZ, HUANG BK, et al. Classification of hippocampus based on microscopic character identification[J].Chin J Marine Drugs, 2021, 40(4):18
[13] 王琪瑞,葛宇清,张光霁, 等. 《中国药典》收载海马药材及其同属近源混伪品的HPLC指纹图谱研究[J].时珍国医国药, 2021, 32(2):349
WANG QR, GE YQ, ZHANG GJ, et al. Study on HPLC fingerprints of hippocampal medicinal materials and their near source adulterants in Chinese Pharmacopoeia[J].Lishizhen Med Mater Med Res, 2021, 32(2):349
[14] 陈信忠,曾韵颖. 海马种类及其鉴定技术分析[J].中国口岸科学技术, 2020(12):48
CHEN XZ, ZENG YY. Analysis of hippocampal species and their identification techniques[J].China Port Sci Technol, 2020(12):48
[15] CAO GY, GENG SX, LUO, Y, et al. The rapid identification of chemical constituents in Fufang Xiling Jiedu capsule, a modern Chinese medicine, by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry and data mining strategy[J].J Sep Sci, 2021, 44(9):1815
[16] 许燕,肖洪贺,段双蕊,等. 动物类中药鉴定技术研究进展[J].中国中医药现代远程教育, 2018, 17:156
XU Y, XIAO HH, DUAN SR, et al. Research progress in identification technology of animal traditional Chinese medicine[J].Chin Med Mod Dist Educ China, 2018, 17:156
[17] 谢薇. 中药材鉴定运用分子标记技术的实践探寻[J].光明中医, 2021, 36(19):3262
XIE W. Practice and exploration of molecular marker technology in the identification of traditional Chinese medicine[J].Guangming J Chin Med, 2021, 36(19):3262
[18] XIONG X, XU WJ, GUO LQ, et al. Development of loop-mediated isothermal amplification(LAMP) assay for rapid screening of skipjack tuna(Katsuwonus pelamis) in processed fish products[J].J Food Compos Anal, 2021, 102:104038
[19] OON JY, KAMARUDIN KR, ISMAIl N, et al. Mitochondrial barcodes of three Malaysian Butterflies originating from Taman Negara Endau Rompin Johor, Malaysia[J].IOP Conf Ser:Earth Environ Sci, 2021, 736:12053
[20] 张云,李炎连,郑炎媚, 等. 基于细胞色素B(Cytb)的市售海蛇药材基原物种的分子鉴定[J].中国中药杂志,2021, 46(5):1102
ZHANG Y, LI YL, ZHENG YM, et al. Identification of Chinese medicine sea snake based on cytochrome B(Cytb)[J].China J Chin Mater Med, 2021, 46(5):1102
