半夏药材炮制品DNA提取方法的优化及分子鉴定
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摘要
目的:优化炮制半夏基因组DNA提取方法并进行市售炮制药材的分子鉴定。方法:以半夏药材炮制品为试验材料,对CTAB水浴后沉淀试剂进行筛选,并优化样品用量、沉淀试剂和裂解时间3个参数,建立半夏炮制品DNA提取方法;采用该法提取市售13份姜半夏和法半夏基因组DNA,用基于ITS2序列的PCR扩增法进行分子鉴定。结果:以CTAB水浴后加入甲醇处理可有效促进DNA沉淀,起始半夏药材0.5 g、水浴裂解时间1 h即可快速高效获得基因组DNA。对所提取DNA样品进行PCR扩增,所有样品全部成功测序。相似性搜索法分析表明,13份样品中10份为掌叶半夏,3份为半夏。结论:该研究建立了简易快速的半夏药材炮制品DNA提取技术,所提DNA满足半夏药材的分子鉴定要求。该结果也提示市售半夏药材混伪情况较多,值得重视。
Objective:To optimize the genomic DNA extraction method for processed drugs of Pinellia ternata and to identify commercial processed drugs via molecular approach.Methods:The processed drugs of Pinellia ternata was used as experimental materials for screening precipitation reagents after CTAB water bath,and optimizing the three parameters of sample amount,precipitation reagent and cracking time,and the extraction method was established.Based on the optimized parameters,the genomic DNAs of 13 commercial processed drugs of Pinellia ternata were extracted and further used for molecular identification via PCR amplification on ITS2 sequence.Results:The addition of methanol after CTAB water bath could efficiently promote the precipitation of DNA,and the starting material amount and water-bath cracking time were determined to be 0.5 g and 1 h,respectively.The extracted DNA samples were amplified by PCR,all samples were sucessfully sequenced.The similarity analysis showed that 10 of 13 commercial processed drugs were Pinellia pedatisecta and another 3 were Pinellia ternata.Conclusion:A simple and rapid method for extracting DNA from processed drugs of Pinellia ternata is established and proves to be suitable for molecular identification on commercial processed drugs of Pinellia ternata.The results also imply that there is a severe situation of adulterants in commercial Pinellia ternata drug.
Objective:To optimize the genomic DNA extraction method for processed drugs of Pinellia ternata and to identify commercial processed drugs via molecular approach.Methods:The processed drugs of Pinellia ternata was used as experimental materials for screening precipitation reagents after CTAB water bath,and optimizing the three parameters of sample amount,precipitation reagent and cracking time,and the extraction method was established.Based on the optimized parameters,the genomic DNAs of 13 commercial processed drugs of Pinellia ternata were extracted and further used for molecular identification via PCR amplification on ITS2 sequence.Results:The addition of methanol after CTAB water bath could efficiently promote the precipitation of DNA,and the starting material amount and water-bath cracking time were determined to be 0.5 g and 1 h,respectively.The extracted DNA samples were amplified by PCR,all samples were sucessfully sequenced.The similarity analysis showed that 10 of 13 commercial processed drugs were Pinellia pedatisecta and another 3 were Pinellia ternata.Conclusion:A simple and rapid method for extracting DNA from processed drugs of Pinellia ternata is established and proves to be suitable for molecular identification on commercial processed drugs of Pinellia ternata.The results also imply that there is a severe situation of adulterants in commercial Pinellia ternata drug.
引文
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[2] 张瑾,谈献和.半夏资源研究进展[J].中国中医药信息杂志,2010,17(5):104-106.
[3] 赵秀清,刘青梅.半夏与几种常见伪品的鉴别[J].山东中医杂志,1997,16(3):131.
[4] 李江维,许润春,王鹏飞,等.半夏曲与伪品水半夏曲的鉴别研究[J].现代中药研究与实践,2016,30(3):16-20.
[5] 陈士林,庞晓慧,姚辉,等.中药DNA条形码鉴定体系及研究方向[J].世界科学技术-中医药现代化,2011,13(5):747-754.
[6] Kumar US,Ratheesh RV,Alexander J,et al.Development of a DNA barcode database of captive animals in Thiruvananthapuram zoo,kerala,India[J].Genome,2015,58(5):239-240.
[7] Kress WJ.Plant DNA barcodes:applications today and in the future[J].Journal of Systematics and Evolution,2017,55(4):291-307.
[8] Badotti F,Oliveira FSD,Garcia CF,et al.Effectiveness of its and sub-regions as DNA barcode markers for the identification of Basidio mycota(Fungi)[J].BMC Microbiology,2017,17(1):42.
[9] 司浩,魏文龙,任伟光,等.基于DNA条形码的半夏属及其伪品分子鉴别[J].植物学报,2014,49(6):710-719.
[10] 冯图,何洋,阮培均.半夏及其易混品的DNA分子鉴定[J].山地农业生物学报,2016,35(2):15-20.
[11] 王琦,孙立立,贾天柱.中药饮片炮制发展回眸[J].中成药,2000,22(1):33-58.
[12] 张雅琴,宋明,孙伟,等.中药材半夏及其混伪品的DNA条形码鉴定研究[J].世界科学技术-中医药现代化,2014,16(8):1725-1729.
[13] 杨俊宝,彭正松,杨军,等.半夏基因组DNA的提取及鉴定[J].现代中药研究与实践,2006,20(3):26-29.
[14] 张景景,祁晓婷,张超,等.ITS2序列鉴定大蓟、小蓟药材及其近缘混伪品[J].世界中医药,2016,11(10):2126-2129.
[15] 张道微,张超凡,董芳,等.甘薯薯块DNA提取方法研究[J].湖南农业科学,2016,(4):5-7.
[16] 涂瑶生,毕晓黎,谭志灿,等.不同炮制品半夏配方颗粒的红外光谱研究[J].广东药学院学报,2013,29(3):266-269.
[17] 程春松,谭天琪,龙泽,等.中成药DNA提取技术优化及其在人参类制剂分子鉴定中的应用[J].中草药,2015,46(17):2549-2555.