阿胶中动物源性DNA提取方法的改进及驴源性成分鉴定

Improvement of Extraction Method of Animal-derived DNA from Colla Corii Asini and Identification of Donkey-Derived Components

目的从高温熬制的阿胶制品中提取微量动物源性DNA,建立并优化聚合酶链反应(PCR)方法快速鉴定阿胶中驴源性成分,建立分子生物学鉴定阿胶品质新方法。方法应用DNA纯化柱替代十二烷基硫酸钠-蛋白酶K(SDS-PK)方法中酚、氯仿等毒性较大的有机溶剂提取阿胶中驴源性基因组DNA,并对SDS-PK方法进行优化。结果提取驴源性基因组DNA最佳阿胶样本量为0.20 g,水浴消化1 h再进行DNA纯化柱处理可快速获得高质量阿胶基因组DNA,纯度均在1.70~1.80之间,可达到(187.8±0.56)ng·μL^-1。应用特异性引物进行PCR扩增、克隆、测序,与Gen Bank已登记的驴物种(MG931481.1)相似性为100%。结论本实验能在90 min内从深加工的阿胶及阿胶制品中提出动物源性基因组DNA片段,提取的DNA纯度和浓度能满足阿胶分子生物学鉴定的要求,建立的PCR方法可快速鉴定阿胶中驴源性成分;克隆得到驴特异性基因片段可作为鉴定阿胶真伪的标准化阳性对照,预期将广泛应用到阿胶及相关制品的质量监督工作中。

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 Gen Bank’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.