development and application of dna extraction methods, pcr parameter optimization and rapid detection reagent for fetus cervi / 中国药学杂志

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.