乌梢蛇及酒乌梢蛇配方颗粒的特异性PCR鉴别研究OA

Objective:To establish a specific PCR method for the identification of Zaocys dhumnades and wine-processed Zaocys dhumnades formula granules,so as to solve the difficulty in accurate identification by traditional methods due to the disappearance of morphological characteristics after processing,and to provide a scientific basis for quality control.Methods:With the mitochondrial 12S rRNA gene of Zaocys dhumnades as the target sequence,specific primers were designed.Genomic DNA was extracted from crude Zaocys dhumnades,wine-processed Zaocys dhumnades and their corresponding formula granules with an optimized DNA extraction method.The specificity,sensitivity and repeatability of the established method were evaluated by PCR amplification,agarose gel electrophoresis and sequencing verification.Results:The designed specific primers amplified a specific band of 347 bp only from crude Zaocys dhumnades,wine-processed Zaocys dhumnades and their formula granules,while no target band was observed in closely related species such as Dinodon rufozonatum,Elaphe taeniura,Elaphe carinata and negative controls.The limit of detection(LOD)for DNA from wine-processed Zaocys dhumnades formula granules was 8.63 ng,and the repeatability test showed satisfactory stability.The sequencing result of the amplified product from formula granules shared 99.8%homology with the 12S rRNA gene sequence of Zaocys dhumnades in GenBank.Conclusion:The established specific PCR identification method exhibits high specificity,sensitivity and stability,which can rapidly and accurately identify Zaocys dhumnades and wine-processed Zaocys dhumnades formula granules,and provides a reliable technical tool for their authenticity identification and quality supervision.