Development and Application of DNA Extraction Methods, PCR Parameter Optimization and Rapid Detection Reagent for Fetus Cervi
ZHOU Ting-ting1, AI Jin-xia1, GAO Li-jun1, SUN Li-yuan1, LI Ming-cheng1,2*, ZHANG Li-hua3
1. School of Medical Technology, Beihua University, Jilin 132013, China; 2. Science and Technology Innovation Center of DNA Fingerprint Detection Technique of Traditional Chinese Medicine, Jilin 132013, China; 3. Leibo Science and Technology Ltd, Jilin 132013, China
Abstract：OBJECTIVE To optimize DNA extraction methods and PCR reaction parameters, and develop an excellent and accurate rapid detection reagent for Fetus Cervi. METHODS The DNA of Fetus Cervi was extracted by the modified salting method, modified SDS method A and modified SDS method B. Four DNA polymerase were chosen from the market and compared with each other. The annealing temperature and annealing time were optimized by classical control variable method and intersected experiment. A rapid detection reagent of Fetus Cervi was developed and then evaluated. RESULTS The A260/A280 ratio of the DNA extracted by modified SDS method B was (1.74±0.05), and the mass concentration was (0.250±0.005)μg·L-1. With high fidelity Taq DNA polymerase, the PCR product concentration could reach (0.185±0.005)μg·L-1. Through these experiments, the annealing temperature was set at 58 ℃ and the annealing time was 30 s. The rapid detection reagent course was established to quickly and accurately identify Fetus Cervi and their artefacts, with one clear and bright band at 563 bp. CONCLUSION The rapid detection reagent of Fetus Cervi combines the optimal DNA extraction method and the optimal PCR reaction parameters, and can accomplish the identification of Fetus Cervi and its pseudo-products with high accuracy and specificity.
周亭亭, 艾金霞, 高丽君, 孙丽媛, 李明成, 张丽华. 鹿胎DNA提取方法、PCR参数优化及其快速检测试剂的开发应用[J]. 中国药学杂志, 2019, 54(18): 1481-1485.
ZHOU Ting-ting, AI Jin-xia, GAO Li-jun, SUN Li-yuan, LI Ming-cheng, ZHANG Li-hua. Development and Application of DNA Extraction Methods, PCR Parameter Optimization and Rapid Detection Reagent for Fetus Cervi. Chinese Pharmaceutical Journal, 2019, 54(18): 1481-1485.