目的 研制川贝母DNA检测试剂盒, 确定试剂盒组成及操作程序, 建立一种简便、快速鉴定川贝母的分子生物学检验方法。方法 分别采用药典法(2010年版《中国药典》增补本)和试剂盒法提取川贝母基因组DNA, 进行PCR反应和RFLP鉴定。结果 采用药典法提取出的川贝母基因组DNA的OD260/OD280值最高为(1.57±0.05), 而采用试剂盒法所提DNA的OD260/OD280值可达(1.73±0.10);两法PCR结果均在300 bp上方有单一明亮条带;RFLP鉴定图谱均在100～250 bp之间出现2条清晰条带。结论 试剂盒法较药典法更稳定, 且核酸提取量及纯度都高于药典法, 2种方法PCR及RFLP鉴定结果完全一致。川贝母DNA检测试剂盒特异性好、灵敏度高、稳定性强, 适用于川贝母的快速检测。
Abstract
OBJECTIVE To develop a kit for detection of DNA of Fritillaria cirrhosa D. Don and optimize its components as well as process protocols, in order to set up a simple, rapid molecular biology method for identification of Fritillaria cirrhosa D. Don. METHODS All genomic DNA of Fritillaria cirrhosa D. Don was extracted by kit assay and pharmacopoeia method recorded in the expanded supplement of China Pharmacopoeia 2010, respectively; ultraviolet spectrophotometer was used to measure the quantity of extracted DNA; PCR amplification and restriction fragment length polymorphism analysis (RFLP) were carried out to identify the authentication of Fritillaria cirrhosa D. Don. RESULTS The maximum value of genomic DNA extracted by pharmacopoeia method was (1.57?0.05)(OD260/OD280) and (1.73?0.10) by kit assay. The PCR amplification showed a single band over 300 bp, while the RFLP showed two distinct bands between 100 and 250 bp in agarose electrophoresis.CONCLUSION The data demonstrated that the kit assay was better than the pharmacopoeia method, especially in the extraction quantity and DNA purity of Fritillaria cirrhosa D. Don nucleic acid; the PCR and RFLP RESULTS showed that the kit assay was consistent with pharmacopoeia method. The detection kit has good specificity, high sensitivity and good stability, so it is suitable for the rapid detection of Fritillaria cirrhosa D. Don.
关键词
川贝母 /
DNA检测试剂盒 /
聚合酶链式反应 /
限制性片段长度多态性分析 /
鉴定图谱
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Key words
Fritillaria cirrhosa D. Don /
DNA detection Kit /
polymerase chain reaction /
restrictive fragment length polymorphism analysis /
RFLP identification atlas
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中图分类号：
R917
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参考文献
[1] TAN Y, ZHANG L H, LI M C, et al. Identification of Chinese herb Fritillaria cirrhosa D. Don by DNA fingerprint . Chin Pharm J(中国药学杂志), 2011, 46 (1):14-16. [2] Ch. P (2010) VolⅠ(中国药典2010年版. 一部). 2010:83-84. [3] LIU Y, ZHANG L H, BO Y. The identification of mitochondrial DNA finger print on the testis et penis Cervi . China Med Pharm (中国医药科学), 2011, 19:112-113. [4] ZHANG L H, LI M C, WANG B M, et al. Identification and characterization of mitochondrial DNA in Martes zibellina L. heart . Chin Pharm J(中国药学杂志), 2008, 43(22):1694-1696. [5] LI M, XIA W, WANG M, et al. Application of molecular genetics method for differentiating Martes zibellina L. heart from its adulterants in traditional Chinese medicine based on mitochondrial cytochromeb gene . Mitochondrial DNA, 2014, 25(1):78-82.[6] LIU T H, WANG J, ZHANG L H. Identification of cytochrome b bene of Chinese herb testudinis carapax et. plastrum . Chin Pharm J(中国药学杂志), 2012, 47(3):182-185. [7] GU Y J, ZHANG L H, FU G L. Mitochondrial DNA fingerprint identification of Velvet antler . Chin Pharm J (中国药学杂志), 2013, 48(3):170-173. [8] WANG S, WANG H, ZHANG L H, et al. Identification of Panax ginsheng C. A. Meyer Cv. Silvatica and Cultivated Panax ginseng by DALP fingerprint . Chin Pharm J (中国药学杂志), 2013, 48(9):677-680. [9] ZHANG T. Establishment the characteristic macrorestriction map of common red-tide algae by RFLP . Qingdao:Ocean University of China, 2009. XU C L, LI H J, LI P, et al. Molecular method for the identification of Bulbus Fritillariae Cirrhosae . J China Pharm Univ (中国药科大学学报), 2010, 41(3):226-230.
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脚注
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基金
吉林省科技发展计划项目(20090906);吉林省战略性新兴产业和高新技术产业发展专项(2013G030);吉林市科技计划发展项目(2013523010)
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