中药材川贝母聚合酶链式反应法-限制性片段长度多态性分析的鉴别及应用研究
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摘要
目的应用中药材川贝母聚合酶链式反应法-限制性片段长度多态性分析的鉴别试剂盒,对北京市、天津市、长春市、吉林市等全国16个城市的70个样品进行真伪鉴定。方法试剂盒法提取川贝母基因组DNA,进行PCR反应和RFLP鉴定,并应用2015年版《中国药典》方法进行比较和复检。结果采用试剂盒法提取出的川贝母基因组DNA纯度良好,OD260/OD280值均在1. 90～2. 1之间,浓度能达到PCR的要求;对70份样品进行检测,正品川贝母37份,伪品33份,伪品率为47. 1%。结论试剂盒法提取核酸的纯度和浓度能满足PCR及RFLP的要求,对全国16个城市川贝母样品进行检测结果表明,市场上川贝母伪品率较高,有关部门应加强对中药市场的质量管理。
OBJECTIVE To identify 70 samples of Fritillariae Cirrhosae Bulbus from 16 cities such as Beijing,Tianjin,Changchun,Jilin and so on by polymerase chain reaction-restriction fragment length polymorphism( PCR-RFLP) analysis kit. METHODS The genomic DNA of Fritillariae Cirrhosae Bulbus was extracted by kit method,and PCR reaction and RFLP identification were carried out. The method of the Chinese Pharmacopoeia of 2015 was used for comparison and re-examination. RESULTS The purity of the genomic DNA of Fritillariae Cirrhosae Bulbus was very good,the OD260/OD280 value was between 1. 90-2. 1,and the concentration could reach the requirement of PCR. Seventy samples were tested,of which 37 were positive and 33 were counterfeit,and the false rate was 47. 1%. CONCLUSION The purity and concentration of the nucleic acid meet the requirements of PCR and RFLP. The test results of the samples from 16 cities show that the counterfeit rate of Fritillariae Cirrhosae Bulbus is high in the market. Relevant government departments should strengthen the quality management of the traditional Chinese medicine market.
OBJECTIVE To identify 70 samples of Fritillariae Cirrhosae Bulbus from 16 cities such as Beijing,Tianjin,Changchun,Jilin and so on by polymerase chain reaction-restriction fragment length polymorphism( PCR-RFLP) analysis kit. METHODS The genomic DNA of Fritillariae Cirrhosae Bulbus was extracted by kit method,and PCR reaction and RFLP identification were carried out. The method of the Chinese Pharmacopoeia of 2015 was used for comparison and re-examination. RESULTS The purity of the genomic DNA of Fritillariae Cirrhosae Bulbus was very good,the OD260/OD280 value was between 1. 90-2. 1,and the concentration could reach the requirement of PCR. Seventy samples were tested,of which 37 were positive and 33 were counterfeit,and the false rate was 47. 1%. CONCLUSION The purity and concentration of the nucleic acid meet the requirements of PCR and RFLP. The test results of the samples from 16 cities show that the counterfeit rate of Fritillariae Cirrhosae Bulbus is high in the market. Relevant government departments should strengthen the quality management of the traditional Chinese medicine market.
引文
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[2]Ch.P(2015)VolⅠ(中国药典2015年版.一部)[S].2015:36.
[3]LIU W,ZHANG W J,LIN L J,et al.Quality analysis of Fritillariae Cirrhosae Bulbus[J].Chin Pharm J(中国药学杂志),2015,50(4):305-309.
[4]Ch.P(2010)VolⅠAddⅠ.(《中国药典》2010年版.第一增补本)[S].2010:83-84.
[5]ZHOU T T,YU W J,LI M C,et al.Development and evaluation of Fritillaria cirrhosa D.Don DNA Detection Kit[J].Chin Pharm J(中国药学杂志),2014,49(6):501-504.
[6]TING T Z,WEN J Y,MINGCHENG L,et al.Development and evaluation of Fritillaria cirrhosa D.Don DNA detection kit[J].Chin Pharm J(中国药学杂志),2014,49(6):501-504.
[7]XU C L,LI H J,LI P,et al.Molecular method for the identification of Bulbus Fritillariae Cirrhosae[J].J China Pharm Univ(中国药科大学学报),2010,41(3):226-230.
[8]ZHANG W J,LIU W,WEI F,et al.Study on identifying adulterants in Fritillariae Cirrhosae Bulbus with polymerase chain reaction-restriction fragment length polymorphisms(PCR-RFLP)method[J].Chin J Pharm Anal(药物分析杂志),2014,34(10):1830-1835.
[9]XU C L,LI H J,LI P,et al.Molecular method for the identification of Bulbus Fritillariae Cirrhosae[J].J China Pharm Univ(中国药科大学学报),2010,41(3):226-230.
[10]WANG W N,CHEN D L,ZHU M F,et al.Nondestructive analysis of Fritillariae Cirrhosae Bulbus by laser Raman spectroscopy[J].Spectrosc Spect Anal(光谱学与光谱分析),2013,33(8):2109-2111.
[11]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[J].Mitochondrial DNA,2014,25(1):78-82.
[12]GU Y J,ZHANG L H,FU G L.Mitochondrial DNA fingerprint identification of Velvet antler[J].Chin Pharm J(中国药学杂志),2013,48(3):170-173.
[13]ZHANG T.Establishment the characteristic macrorestriction map of common red-tide algae by RFLP[D].Qingdao:Ocean University of China,2009.
[14]XU C L,LI H J,LI P,et al.Molecular method for the identification of Bulbus Fritillariae Cirrhosae[J].J China Pharm Univ(中国药科大学学报),2010,41(3):226-230.