蕲蛇标准品DNA特异鉴定指纹片段的克隆及评价  

Evaluation on Cloning of Specific DNA Fingerprinting Fragments of Agkistrodon acutu s Guenther Standard Samples

作　　者：赵万韬 包学英[1] 李明成 ZHAO Wantao;BAO Xueying;LI Mingcheng(Affiliated Hospital of Beihua University,Jilin 132011,China;Innovation Center for Detection Technology on DNA Fingerprint of Traditional Chinese Medicine,Jilin 132013,China)

机构地区：[1]北华大学附属医院,吉林吉林132011 [2]吉林省中药DNA指纹检测技术科技创新中心,吉林吉林132013

出　　处：《北华大学学报（自然科学版）》2022年第1期75-78,共4页Journal of Beihua University（Natural Science）

基　　金：吉林省科技创新中心建设项目(20190902018TC).

摘　　要：目的以工程菌为载体,寻找可以长期保存中药材蕲蛇标准品特异DNA指纹区段的克隆技术.方法采用蕲蛇DNA检测试剂盒提取蕲蛇标准品基因组DNA,按照2010年版《中国药典》收录的方法进行蕲蛇特异DNA片段PCR扩增,将PCR产物与质粒载体重组后导入工程菌细胞内;体外培养细菌,提取重组质粒,应用PCR及测序方法鉴定插入片段是否正确.将鉴定正确、携带正品蕲蛇特异DNA序列的菌落置-80℃冰柜保存,分别以30 d、60 d、3个月和6个月为时间点进行复苏,进行稳定性评价.结果PCR扩增蕲蛇标准品特异DNA片段长度约为300 bp,插入质粒载体后,转入工程菌细胞内的蕲蛇DNA经体外克隆、PCR扩增及测序鉴定,扩增产物与目标大小一致,序列与Genbank中蕲蛇序均一致.将4个时间点复苏后的菌落进行PCR扩增后,条带清晰,没有降解.结论采用分子克隆技术保存蕲蛇特异基因片段的方法可行、有效,方法简单明了,获得的标准品DNA稳定性良好,结果可靠.Objective To establish a method for effective preservation of DNA fingerprint fragments of Agkistrodon acutu s Guenther standard samples.Method Genomic DNA was extracted by Agkistrodon acutu s Guenther DNA detection kit and then introduced into biological host cells.A large number of Agkistrodon acutu s Guenther specific DNA clones were produced by host cell self-replication.Recombinant plasmids were extracted by plasmid extraction kit,and PCR and sequencing methods were used to check and identify whether the inserts were correct.Colonies carrying authentic developed by the team,and Agkistrodon acutu s Guenther specific DNA fragments were cloned,combined with plasmid vector,Agkistrodon acutu s Guenther specific DNA sequences were stored in-80℃freezer and resuscitated at time points of 30 d,60 d,3 months,and 6 months for stability studies.Results After PCR amplification of Agkistrodon acutu s Guenther DNA clones transferred into cells,the agarose gel electrophoresis illustrated that the patterns were clear,and the results of multiple tests were consistent.After PCR amplification of the recovered colonies at the four time points,the bands were bright and clear in the agarose gel electrophoresis assay.Conclusion The method of preserving Agkistrodon acutu s Guenther specific gene fragments by molecular cloning technique is feasible,effective and stable,and can be used as an effective basis for establishing a database for DNA fingerprinting identification of traditional Chinese medicine.The method is simple,clear and the results are reliable.

关 键 词：蕲蛇标准品 DNA指纹鉴定技术 分子克隆 PCR扩增 

分 类 号：R927[医药卫生—药学] R932