4种动物源性成分多重real-time PCR检测方法的建立及其在驴肉制品检测中的应用  

Establishment and Application of Multiplex Real-Time PCR Method for Rapid Detection of Four Animal-Derived Components in Donkey Meat Products

作　　者：范维[1] 高晓月[1] 李贺楠[1] 董雨馨 刘虹宇 李宇轩 郭文萍[1] FAN Wei;GAO Xiaoyue;LI Henan;DONG Yuxin;LIU Hongyu;LI Yuxuan;GUO Wenping(China Meat Research Center,Beijing Academy of Food Science,Beijing 100068,China)

机构地区：[1]中国肉类食品综合研究中心,北京食品科学研究院,北京100068

出　　处：《食品科学》2023年第8期317-323,共7页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2018YFC1603400);北京市优秀人才培养资助青年骨干个人项目(2017754154700G101)。

摘　　要：建立一种同时快速检测驴、马、猪及鸭源性成分的四重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以4种源性成分的Nad5、ATpase6、ATP8、cytb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,并对该方法进行方法学验证,同时对不同掺入比例模拟样品、不同加工工艺模拟样品和实际驴肉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。当Ct值≤35.0时,方法对16种非目标源性具有良好特异性;灵敏度可检测到质量浓度为2×10^(-4)ng/μL的模板DNA;对生肉的检出限为肉含量的0.001%,对熟肉制品的检出限为肉含量的0.01%;对100份实际样品进行检测,结果与标准方法一致,说明建立的多重real-time PCR法可用于肉及肉制品中常见掺假源性成分的检测。A multiplex real-time polymerase chain reaction(PCR)method for simultaneous rapid detection of donkey-,horse-,pig-and duck-derived components was established.For this purpose,the Nad5,ATpase6,ATP8 and cytb were used as target genes to design specific primers and TaqMan probes,and the 18S rRNA gene was used as internal reference gene.The developed method was validated and applied to detect simulated adulterated meat products,simulated processed meat products and actual donkey meat samples.This method was characterized by high-throughput,and excellent sensitivity and specificity.The system had no cross-reaction to non-target sources when the cycle threshold(Ct)value did not exceed 35.0.The sensitivity was 2×10^(-4) ng/μL of template DNA.The detection limit for raw and cooked meat was 0.001%and 0.01%of the meat content,respectively.The results of this method for 100 actual samples were consistent with those of the national standard method,indicating that the multiplex real-time PCR method can be used for the detection of commonly adulterants in meat and meat products.

关 键 词：多重实时聚合酶链式反应 掺假鉴别 驴肉 马肉 猪肉 鸭肉 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程]