Improvement of Extraction Method of Animal-derived DNA from Colla Corii Asini and Identification of Donkey-Derived Components
CHEN Si-xiu1a, ZHANG Xin-fang1a, LIU Wen-yan1a, WANG Tian-tian1a, ZHAO Yun-dong1a, WANG Yan-shuang1b, GAO Li-jun1a, LI Ming-cheng1a,2, SUN Li-yuan1a*
1a. School of Laboratory Medical;1b. School of Medical, Beihua University, Jilin 132013, China; 2. Innovation Center of DNA Fingerprint Detection Technology of Traditional Chinese Medicine, Jilin 132013, China
Abstract：OBJECTIVE To extract microamounts of animal-derived DNA from the products of Colla Corii Asini boiled at high temperature, establish and optimize a rapid identification method of donkey-derived components in Colla Corii Asini by polymerase chain reaction(PCR), and establish a new molecular biological method for assessing the quality of Colla Corii Asini.METHODS The donkey-derived genomic DNA was extracted by DNA purification column instead of phenol, chloroform and other toxic organic solvents in SDS-PK method, and the SDS-PK method was optimized with the donkey-derived genomic DNA.RESULTS The optimum sample size of Colla Corii Asini was 0.20 g. High quality genomic DNA of Colla Corii Asini could be obtained quickly after digestion in water bath for 1 h and then purified by DNA purification column. The purity ranged from 1.70 to 1.80, and the concentration of Colla Corii Asini could reach (187.8±0.56)ng·μL-1. PCR amplification, cloning, and sequencing were performed using specific primers, and the similarity to GenBank′s registered Donkey species (MG931481.1) was 100%.CONCLUSION This study provides animal-derived genomic DNA fragments from deep-processed Colla Corii Asini and Colla Corii Asini products within 90 min. The purity and concentration of extracted DNA can meet the requirements of molecular biological identification of Colla Corii Asini. The established PCR method can quickly identify the scorpion-derived components in Colla Corii Asini. The cloned donkey specific gene fragment can be used as a standard positive control to identify the authenticity of Colla Corii Asini. It is expected that it will be widely used in the quality supervision of Colla Corii Asini and related products.
陈思秀, 张馨方, 刘玟妍, 王天添, 赵云冬, 王艳双, 高丽君, 李明成, 孙丽媛. 阿胶中动物源性DNA提取方法的改进及驴源性成分鉴定[J]. 中国药学杂志, 2019, 54(22): 1840-1845.
CHEN Si-xiu, ZHANG Xin-fang, LIU Wen-yan, WANG Tian-tian, ZHAO Yun-dong, WANG Yan-shuang, GAO Li-jun, LI Ming-cheng, SUN Li-yuan. Improvement of Extraction Method of Animal-derived DNA from Colla Corii Asini and Identification of Donkey-Derived Components. Chinese Pharmaceutical Journal, 2019, 54(22): 1840-1845.
JIANG Y P,DI Z Q, WANG Y T, et al. Study on anti-fatigue, anti-oxidative and hemostatic effects of small molecule Asini Corii Colla[J/OL]. Chin Pharmacol Bull(中国药理学通报), 2019, 35(2):203-208.
DI Z Q, HU J F, ZHANG L, et al. Study on hematopoiesis, antifatigue and hemostatic effects of Asini Corii Colla[J]. Drug Eval Res(药物评价研究), 2018, 41(4):562-566.
HUANG Y L, HUANG H Y, ZHANG H Y, et al. Effect of Shenling donkey hide gelatin compound plaster on anti fatigue effect of mice with Qi deficiency constitution[J]. Lishizhen Med Mater Med Res(时珍国医国药),2016,27(2):346-347.
ZHANG L, ZHU H F, CHEN H H, et al. Anti-fatigue effect of Fufang Ejiao Jiang on mice[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志), 2013, 19(19):254-257.
DI Z Q, JIANG Y P, WANG Y T, et al. Effects of small molecular Asini Corii Colla on immune function in mice[J].Drug Eval Res(药物评价研究), 2018, 41(9):1602-1605,1667.
CHEN S X, LIU W Y, GAO Q Y, et al. Development and evaluation of donkey-derived DNA detection kit[J].Chin J Anim Sci(中国畜牧杂志), 2018, 54(8):121-125.
ZHANG J Z. Authentic identification of Ejiao[J]. For All Health(大家健康:学术版), 2013, 7(14):32.
DU Y B, FAN H R, YAN Z. Research progress on chemical constituents and pharmacological action of Ejiao[J].J Tianjin Med Univ(天津医科大学学报), 2018, 24(3):267-270.
ZHU L L, DOU D Q.Determination of protein and amino acid content in donkey-hide gelatin and deer skin glue[J]. Asia-Pacific Tradit Med(亚太传统医药), 2018, 14(6):48-50.
GB 5009.5-2016(食品安全国家标准食品中蛋白质的测定) [S]. 2016.
LUO M H, XIAO B Y, NIE P, et al. Application of DNA molecular identification method to distinguish donkey-derived components in Ejiao[J]. Chin J Pharm Anal(药物分析杂志), 2017, 37(2):202-211.
ZHANG Q F, LIU Y Y, BIAN R R, et al. New method for extraction of DNA from donkey hide gelatin and its prepartions[J]. Food Drug(食品与药品), 2015, 17(6):396-399.
CHEN Z X, GONG L G, QIAN Y K.Improvement of extraction method of DNA from Asini Corii Colla[J]. Chin J Exp Tradit Med Form(中国实验方剂学杂志), 2014, 20(21):13-16.
LV P, ZHOU X S, YOU J H, et al. Extraction of trace amount of severely degraded DNA[J]. Z Naturforsch C, 2009, 64(7-8):581-589.