位点特异性PCR鉴别水牛角及其伪品  

Site-Specific PCR for Distinguishing Bubali Cornu from Adulterants

作　　者：张竞研 谭冰艳 康安[1] 王协和 李松[2] 陈盛君[2] 刘睿[1] 段金廒[1] ZHANG Jing-yan;TAN Bing-yan;KANG An;WANG Xie-he;LI Song;CHEN Sheng-jun;LIU Rui;DUAN Jin-ao(Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine,National Administration of Traditional Chinese Medicine Key Laboratory for Chinese Medicine Resources Recycling Utilization,Nanjing University of Chinese Medicine,Nanjing 210023,China;Tianjiang Pharmaceutical Co.,Ltd.,Wuxi 214429,China)

机构地区：[1]南京中医药大学/江苏省中药资源产业化过程协同创新中心/中药资源产业化与方剂创新药物国家地方联合工程研究中心/国家中医药管理局中药资源循环利用重点研究室,江苏南京210023 [2]江阴天江药业有限公司,江苏无锡214429

出　　处：《中国现代中药》2024年第9期1462-1469,共8页Modern Chinese Medicine

基　　金：国家自然科学基金面上项目(81973450)。

摘　　要：目的:开发一种针对水牛角的特异性聚合酶链式反应(PCR)鉴别方法,快速、准确地鉴别水牛角与其他混伪品。方法:通过比对水牛、黄牛、牦牛、山羊等动物的细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列差异,筛选水牛的特异性单核苷酸多态性(SNP)位点,设计出相应的鉴别引物,并对影响PCR体系的相关条件进行优化,考察验证方法的耐受性和适用性。结果:PCR条件为扩增体系20μL、退火温度60℃、循环次数32次、DNA模板量为100 ng、10μmol·L^(-1)正反引物对各0.8μL时,使用设计的鉴别引物SN1对水牛角进行PCR扩增,产物经琼脂糖凝胶电泳后出现约358 bp的特异性条带,其他物种的样品均无条带出现。结论:该PCR鉴别方法特异性好,能迅速、准确地鉴别水牛角和其他常见动物角类样品,为水牛角的真伪鉴别提供参考。Objective:To develop a polymerase chain reaction(PCR)-based method for the rapid and accurate distinguishing of Bubali Cornu from adulterants.Methods:The specific single nucleotide polymorphisms(SNPs)of Bubalus bubalis Linnaeus were screened by comparison on the cytochrome C oxidase subunitⅠ(COⅠ)gene sequences between Bubalus bubalis,Bos taurus,Bos mutus,and Capra hircus,on the basis of which corresponding primers were designed.The PCR system was optimized,and the tolerability and applicability of the method were evaluated.Results:The PCR conditions were as follows:the amplification system of 20μL,annealing temperature of 60℃,32 cycles,DNA template of 100 ng,0.8μL forward and reverse primers(10μmol·L^(-1)each).The amplification of Bubali Cornu samples was carried out with the designed primer SN1,which yielded a specific band at approximately 358 bp after agarose gel electrophoresis,while the samples from other species did not show any band.Conclusion:The developed PCR method enables rapid and accurate distinguishing of Bubali Cornu from other common animal horn samples.This method serves as a basis for distinguishing Bubali Cornu from adulterants.

关 键 词：水牛角 聚合酶链式反应 鉴别 

分 类 号：R282[医药卫生—中药学]