可视化核酸等温扩增方法快速检测羊肉源性成分  

Visual detection of mutton-derived components by isothermal amplification method

作　　者：吴晓芳[1] 杨海兵 朱晓娟[1] 纪蕾[1] 徐德顺[1] 徐昌平[2] WU Xiao-fang;YANG Hai-bing;ZHU Xiao-juan;JI Lei;XU De-shun;XU Chang-ping(Huzhou Municipal Center for Disease Control and Prevention,Huzhou,Zhejiang 313000,China;不详)

机构地区：[1]湖州市疾病预防控制中心,浙江湖州313000 [2]浙江省疾病预防控制中心,浙江杭州310051

出　　处：《中国卫生检验杂志》2021年第7期817-819,822,共4页Chinese Journal of Health Laboratory Technology

基　　金：湖州市科技计划项目(2018GY23)。

摘　　要：目的建立羊肉源性成分核酸等温扩增可视化快速检测方法。方法用加热煮沸方法直接提取羊肉标本中的DNA;根据羊属特异性的Cytb基因序列的片段设计扩增引物,优化反应体系和反应条件,确立最佳等温扩增温度和扩增时间,对羊肉制品进行可视化等温扩增。并将建立的核酸等温扩增方法与荧光定量PCR法进行实际样品的检测。结果建立的可视化核酸等温扩增快速检测方法最佳的等温扩增温度和扩增时间为65℃30 min;该方法的最低检测限为1μg;对肉制品中目标检测物具有高度特异性。本研究对实际样品的检测结果与荧光PCR方法一致。结论该方法用于羊肉源性成分快速检测,具有操作简便、敏感性高、特异性强以及检测结果可肉眼观察等特点,适用于在超市、农贸市场进行羊肉类成分的现场快速检测。Objective To establish a visual and rapid detection technique for mutton-derived components by isothermal amplification.Methods The DNA in mutton specimens was directly extracted by heating and boiling method.Amplification primers were designed based on the Cytb gene sequence specific to the genus of sheep,optimize the reaction system and reaction conditions,establish the optimal isothermal amplification temperature and amplification time to achieve mutton products are visually isothermally amplified.The newly established nucleic acid isothermal amplification method and fluorescence quantitative PCR method were used to detect actual samples.Results The established rapid visual isothermal amplification detection method can be completed at 65℃for 30 min.The minimum detection limit of this method was 1μg.It has high specificity for the target detection object in meat products.The detection results of the actual samples in this study are consistent with the fluorescent PCR method.Conclusion The established method provides a useful tool for rapid detection of mutton-derived components.It has the advantages of simple operation,high sensitivity and specificity,observing the reaction results by the naked eye,which is suitable for the rapid detection of mutton-derived components in supermarkets and farmers markets.

关 键 词：核酸等温扩增 可视化检测 羊肉源性成分 

分 类 号：R155[医药卫生—营养与食品卫生学]