基于mt DNA cyt b基因兔睾丸鉴定方法的建立及检测试剂盒的研制  

Establishment of a rabbit testis identification method based on mt DNA cyt b gene and the development of its detection kit

作　　者：王艳双[1,3] 姜海瀛 李坦诚 艾金霞 高丽君 李明成 张丽华[5] 孙丽媛 WANG Yan-shuang;JIANG Hai-ying;LI Tan-cheng;AI Jin-xia;GAO Li-jun;LI Ming-cheng;ZHANG Li-hua;SUN Li-yuan(Beihua University Medical School Jilin 132013,China;Beihua University Medical Technology school,Jilin 132013,China;Science and Technology Innovation Center of Jilin Province Traditional Chinese Medicine DNA Fingerprint Detection Technology,Beihua University,Jilin 132013,China;Changchun Institute of Food and Drug Inspection,Changchun 130000,China;Leibo Science and Technology Ltd.in Jilin,Jilin 132013,China)

机构地区：[1]北华大学医学院,吉林132013 [2]北华大学医学技术学院,吉林132013 [3]北华大学吉林省中药DNA指纹检测技术科技创新中心,吉林132013 [4]长春市食品药品检验研究所,长春130000 [5]吉林雷博科技有限公司,吉林132013

出　　处：《药物分析杂志》2020年第8期1376-1383,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：吉林省科技发展计划项目(20180201023YY,20190303093SF,20190301014NY,20200404152YY,20200403047SF);吉林省发改委项目(2018C048-2);吉林省科技创新中心建设项目(20190902018TC)。

摘　　要：目的:建立兔睾丸DNA指纹鉴定方法,研发出兔睾丸DNA检测试剂盒。方法:以穴兔线粒体DNA(mt DNA)细胞色素b(cyt b)基因作为靶基因,设计兔睾丸特异性引物,利用现代DNA指纹技术,研制兔睾丸DNA提取及检测试剂,并对试剂的特异性、重复性及稳定性进行考察;应用分子克隆及测序技术,克隆兔睾丸DNA检测的标准品;进而建立兔睾丸DNA指纹鉴定方法,制定质量标准,开发出兔睾丸DNA检测试剂盒。结果:自主研发的试剂提取的兔睾丸DNA结构完整性较好,DNA浓度与纯度均较高;59℃时引物的特异性最强;兔睾丸正品均在326 bp处出现1条特异性扩增条带,而阴性及空白对照均未出现扩增条带;克隆测序后的兔睾丸DNA序列与穴兔mt DNA特异指纹区段同源性100%。结论:本研究建立的兔睾丸DNA指纹鉴定方法特异、准确、可靠,研发的DNA检测试剂盒操作简便,结果稳定,适于普及推广使用。Objective:To establish a DNA fingerprint identification method for rabbit testis and develop a DNA detection kit for rabbit testis. Methods:In this study,the cave rabbit mitochondrial DNA(mt DNA) cytochrome b(cyt b) gene was used as the target gene to design the specific primers of rabbit testis,the extraction and detection reagents of rabbit testis DNA were developed using DNA fingerprinting technique,and the specificity,reproducibility,and stability of the reagents were investigated. Molecular cloning and sequencing technique were used to clone the standard substance of rabbit testis DNA detection,then a DNA fingerprint detection method of rabbit testis and the quality standard for rabbit testis were established,and a DNA detection kit of rabbit testis was developed. Results:The structure of DNA extracted from rabbit testis by self-developed reagents showed good structural integrity,and both the concentration and purity of DNA were high. The specificity of primers was the strongest at 59 ℃. A specific amplification band of the original rabbit testis samples was found at 326 bp,but no amplification band was found in the negative and blank control groups. The DNA sequence of rabbit testis after cloning and sequencing was 100% homologous to the mt DNA specific fingerprint region of cave rabbit. Conclusion:The DNA fingerprint identification method of rabbit testis established in this study is specific,accurate and reliable. The developed DNA detection kit is easy to use and showing stable results. It is suitable for popularization and application.

关 键 词：兔睾丸 线粒体DNA细胞色素b基因 DNA指纹技术 PCR技术 分子克隆 基因测序 DNA检测试剂盒 

分 类 号：R917[医药卫生—药物分析学]