基于物种特异性PCR方法的鸡内金真伪鉴别
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摘要
目的:建立鉴别鸡内金及其常见伪品的物种特异性聚合酶链式反应(PCR)方法,对全国中药材及饮片专项抽验任务中所抽取的鸡内金饮片进行真伪鉴别,评价市场上鸡内金的真伪情况。方法:根据鸡、鸭、鹅的12S序列差异,设计或优化物种特异性PCR鉴别引物,对影响PCR结果的主要因素退火温度,PCR循环次数,模板DNA浓度,Taq酶种类等进行方法学考察和优化。使用优化的特异性PCR方法对鸡、鸭、鹅内金样品进行DNA分子鉴别。结果:在进行方法学考察确定最优鉴别条件的基础上,当PCR退火温度为55℃,循环次数为30次的条件下,当使用文中筛选得到的鸡物种特异性鉴别引物时,所测鸡内金饮片均检出约为273 bp的特异性扩增条带,混伪品鸭内金和鹅内金均无相应扩增条带;当使用鸭和鹅鉴别引物时,均未检出相应的扩增条带。结论:位点特异性PCR方法能够快速准确的鉴别出鸡内金真伪品,可用于全国中药材及饮片专项抽验任务中鸡内金的真伪鉴定,目前市场上用鸭内金和鹅内金充鸡内金现象较少,质量评价较好。
Objective:To screen the specific reverse primers of Galli Gigerii Endothelium Corneum,duck gizzard membrane and goose gizzard membrane,and establish a specific PCR for molecular identifying Galli Gigerii Endothelium Corneum and its common adulterants.Method:Based on the mutation sites on the 12 S rRNA sequence,specific polymerase Chain reaction(PCR) identify primers were designed for chicken,duck and goose gizzard membrane.The specific PCR reaction conditions were optimized,and the PCR identification method was explored and verified in terms of tolerance and feasibility.Thirty batches of Galli Gigerii Endothelium Corneum decoction pieces extracted from the test were identified.Result:Thirty batches of Galli Gigerii Endothelium Corneum decoction pieces were detected using chicken-specific primers,273 bp of specific bands was amplified and visualized on the agarose electrophoregram.When duck and goose primers were used,no corresponding amplified band was detected.Conclusion:The allele-specific PCR method can be used as a rapid and accurate method to identify Galli Gigerii Endothelium Corneum.It is a promise method for special sampling tasks of Chinese herbal medicine and decoction tablets nationwide.
Objective:To screen the specific reverse primers of Galli Gigerii Endothelium Corneum,duck gizzard membrane and goose gizzard membrane,and establish a specific PCR for molecular identifying Galli Gigerii Endothelium Corneum and its common adulterants.Method:Based on the mutation sites on the 12 S rRNA sequence,specific polymerase Chain reaction(PCR) identify primers were designed for chicken,duck and goose gizzard membrane.The specific PCR reaction conditions were optimized,and the PCR identification method was explored and verified in terms of tolerance and feasibility.Thirty batches of Galli Gigerii Endothelium Corneum decoction pieces extracted from the test were identified.Result:Thirty batches of Galli Gigerii Endothelium Corneum decoction pieces were detected using chicken-specific primers,273 bp of specific bands was amplified and visualized on the agarose electrophoregram.When duck and goose primers were used,no corresponding amplified band was detected.Conclusion:The allele-specific PCR method can be used as a rapid and accurate method to identify Galli Gigerii Endothelium Corneum.It is a promise method for special sampling tasks of Chinese herbal medicine and decoction tablets nationwide.
引文
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[9]许翊,陆妍,武海萍,等.蛇类中药饮片分子鉴别方法的研究进展[J].中国中药杂志,2017,42(15):2930-2933.
[10]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,2018,24(15):57-64.
[11]马德源,谭晴晴,陈力群,等.中药材五灵脂特异性PCR鉴别方法的研究[J].食品与药品,2018,20(6):413-417.
[12]Rodríguez M A,García T,González I,et al.Identification of goose,mule duck,chicken,turkey,and swine in foie gras by species-specific polymerase chain reaction[J].J Agri Food Chem,2003,51(6):1524-1529.
[2]汪岩,王月珍,马千里,等.中药鸡内金研究进展[J]中国民族民间医药,2014(19):10-12.
[3]郑雁,苗明三.鸡内金的现代研究特点分析[J].中医学报,2015,30(12):1796-1797.
[4]戴笑盈,王宝庆,罗梦琪,等.鸡内金检测方法的研究进展[J].黑龙江科技信息,2017(13):63-64.
[5]金伶佳.鸡内金炮制工艺及质量标准规范化研究[D].沈阳:辽宁中医药大学,2011.
[6]吕武清,马珠.鸭、鹅、鸡内金化学成分比较研究[J].中药材,1992,15(1):14-16.
[7]陈士林.中药DNA条形码分子鉴定[M].北京:人民卫生出版社,2012:398.
[8]曲萌,崔继春,董志恒,等.鸡内金的分子鉴定研究[J].中国中药杂志,2009,34(24):3192-3194.
[9]许翊,陆妍,武海萍,等.蛇类中药饮片分子鉴别方法的研究进展[J].中国中药杂志,2017,42(15):2930-2933.
[10]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,2018,24(15):57-64.
[11]马德源,谭晴晴,陈力群,等.中药材五灵脂特异性PCR鉴别方法的研究[J].食品与药品,2018,20(6):413-417.
[12]Rodríguez M A,García T,González I,et al.Identification of goose,mule duck,chicken,turkey,and swine in foie gras by species-specific polymerase chain reaction[J].J Agri Food Chem,2003,51(6):1524-1529.