基于聚合酶链式反应⁃限制性内切酶长度多态性方法鉴别鹿角(马鹿)药材及其配方颗粒  

Identification of Cervi Cornu(Cervus elaphus)and its formula granules based on PCR⁃RFLP

作　　者：周海琴 海丰 胡剑虹 李松[1] 陈盛君[1] 张开雪 程梦娟 ZHOU Hai-qin;HAI Feng;HU Jian-hong;LI Song;CHEN Sheng-jun;ZHANG Kai-xue;CHENG Meng-juan(Jiangyin Tianjiang Pharmaceutical Co.,Ltd.,Jiangyin 214434,China)

机构地区：[1]江阴天江药业有限公司,江苏江阴214434

出　　处：《中国中药杂志》2024年第6期1517-1525,共9页China Journal of Chinese Materia Medica

基　　金：江苏省科技成果转化专项(BA2021018)。

摘　　要：鹿角为鹿科马鹿Cervus elaphus或梅花鹿C.nippon已骨化的角或锯茸后翌年春季脱落的角基。目前市场上鹿角基原混杂,为快速准确地鉴别鹿角多基原及混伪品,研究通过比较马鹿、梅花鹿及其混伪品线粒体条形码Cytb基因序列差异,筛选获得鹿角的特异性单核苷酸多态性(SNP)位点,设计鹿角特异性鉴别引物dishmy⁃F及dishmy⁃R,分别采集鹿角及其混伪品,建立鹿角配方颗粒特异性PCR鉴别方法并优化PCR反应体系,对影响该方法重复性的Taq酶种类、PCR仪型号进行耐受性和适用性考察,再使用限制性内切酶MseⅠ对梅花鹿与马鹿的扩增产物进行酶切。结果显示,当退火温度为56℃,循环次数为35时,鹿角药材、标准汤剂及其配方颗粒经PCR反应及凝胶电泳后,马鹿可在近100 bp处观察到单一明亮的特异性鉴别条带,且马鹿的条带较梅花鹿短约60 bp,而其他混伪品如驼鹿、驯鹿、白尾鹿、黑尾鹿、狍子、白唇鹿等及阴性对照均无条带。该研究建立的PCR⁃RFLP方法可快速准确的鉴别出鹿角药材、标准汤剂和配方颗粒中的马鹿成分,并可区分梅花鹿和其他混伪品,同时为其他中药配方颗粒质量标准研究提供了理论依据。Cervi Cornu is the ossified antler,or the base antler that falls off in the spring of the following year after the pilose antler is sawn off from Cervus elaphus or C.nippon,as a precious traditional Chinese medicine,has been recognized for its medicinal value and widely used in clinical practice.However,the origins of Cervi Cornu are miscellaneous,and Cervi Cornu is even mixed with adul⁃terants in the market.Currently,there is a shortage of ways to identify Cervi Cornu and no standard to control the quality of Cervi Cor⁃nu.So it is valuable to develop a way to effectively identify Cervi Cornu from the adulterants.In this study,the differences in the mito⁃chondrial barcode cytochrome b(Cytb)gene sequences of C.elaphus,C.nippon and their related species were compared and the spe⁃cific single nucleotide polymorphism(SNP)sites on the Cytb sequences of Cervi Cornu were screened out.According to the screened SNPs,Cervi Cornu⁃specific primers dishmy⁃F and dishmy⁃R were designed.The PCR system was established and optimized,and the tolerance and feasibility of Taq polymerases and PCR systems affecting the repeatability of the PCR method were investigated.The am⁃plification products of C.elaphus and C.nippon were digested using the restriction enzyme MseⅠ.The results showed that after elec⁃trophoresis of the product from PCR with the annealing temperature of 56℃and 35 cycles,a single specific band at about 100 bp was observed for C.elaphus samples,and the product of C.elaphus samples was 60 bp shorter than that of C.nippon samples.There was no band for adulterants from other similar species such as Alces alces,Rangifer tarandus,Odocoileus virginianus,O.hemionus,Cap⁃reolus pygargus,Przewalskium albirostis and negative controls.The polymerase chain reaction⁃restriction fragment length polymorphism(PCR⁃RFLP)method established in this study can quickly and accurately identify Cervi Cornu originated from C.elaphus in crude drugs,standard decoctions,and formula granules,and distinguish the origins of

关 键 词：鹿角 聚合酶链式反应⁃限制性内切酶长度多态性方法(PCR⁃RFLP) 分子鉴别 

分 类 号：R282.5[医药卫生—中药学]