摘要
目的:为保障临床用药的安全性和有效性,建立地龙(参环毛蚓)药材及其加工制品混伪、掺假特异性聚合酶链式反应(PCR)鉴定方法。方法:根据地龙氧化酶亚基1(COⅠ)基因序列筛选出地龙配方颗粒的稳定引物区间,并根据区间内筛选获得地龙的特异性单核苷酸多态性(SNP)位点,根据SNP位点设计地龙特异性鉴别引物dlshmy-F及dlshmy-R,分别采集地龙及其混伪品,建立地龙配方颗粒特异性PCR鉴别方法并优化PCR反应体系,对该方法进行耐受性和适用性考察。结果:退火温度为62℃,循环次数为36次,地龙及其配方颗粒经PCR反应及凝胶电泳后,可在约170 bp处观察到单一明亮的特异性鉴别条带,其他近源伪品如通俗环毛蚓、保宁腔蚓、栉盲环毛蚓等20种伪品及阴性对照均无条带。结论:该研究建立的特异性PCR鉴别方法可快速准确的鉴别出地龙药材、配方颗粒、冻干粉中的参环毛蚓源性成分,准确鉴定出全国广地龙中药材及饮片以及地龙的基原,也可为其他的中药配方颗粒质量标准研究提供了参考。
Objective:In order to ensure the safety and effectiveness of clinical drug use,the identification method of mixed and adulterated specific polymerase chain reaction(PCR)identification of Pheretima aspergillum and its processed products was established.Method:Based on the cytochrome C oxidase subunit I sequence of P.aspergillum,primers were designed to cover the whole sequences,and the stable DNA ranges suitable for the identification of Pheretima(P.aspergillum)formula granule were screened out.Specific primers were designed according to the specific single nucleotide polymorphisms(SNP)of P.aspergillum in the stable DNA range.The P.aspergillum and its mixture were collected respectively,the PCR reaction system was established and optimized,and PCR reaction system and procedure were optimized,and the tolerance and applicability were investigated.Result:When the annealing temperature was 62 ℃ and the cycle number was36,both P.aspergillum formula granule and its formula particles could amplify a single specific identification band of about 170 bp,and the other 20 adulterants and negative controls had no band.Conclusion:The allelespecific PCR identification method established in this study can quickly and accurately identify the P.aspergillum formula granule.The orgin of Chinese herbal medicine and decoction pieces and P.aspergillum were accurately identified.It can also provide a reference for other studies on the quality standard research of other Chinese herbal formula granule.
作者
胡力
赵玉洋
袁媛
蒋超
HU Li;ZHAO Yuyang;YUAN Yuan;JIANG Chao(School of Pharmacy,Anhui University of Traditional Chinese Medicine,Hefei 230012,China;State Key Laboratory Breeding Base of Dao-Di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第17期119-126,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
中国中医科学院科技创新工程重大攻关项目(CI2021A04104)
东城区优秀人才培养项目(2021-dchrcpyzz-7)
青年岐黄学者项目。
