摘要
目的建立快速检测牛肉源性成分的环介导等温扩增(LAMP)-核酸试纸条方法。方法用直接加热煮沸方法提取鲜肉标本中的DNA;根据牛属特异性的16S基因序列片段设计引物和探针,优化扩增温度和扩增时间;结合核酸试纸条显色,将建立的快速检测方法与商品化的荧光定量PCR试剂盒比较。结果 LAMP检测方法的最佳反应条件为65℃30 min,5 min内通过扩增产物显色肉眼即可判断结果;具有高度特异性,只有牛肉标本出现特异性扩增;最低检测限为10μg;与荧光PCR方法比较,两者检测结果一致。结论环介导等温扩增-核酸试纸条检测方法具有提取核酸方便、高效、敏感、检测时间短以及防污染等优点,非常适用在基层检验机构以及农贸市场、超市的现场快速检测。
Objective To establish a rapid detection method of beef derived components by loop-mediated isothermal amplification combining nucleic acid test strip.Methods DNA in fresh meat samples was extracted by direct heating and boiling.Specific primers and probes were designed according to the specific 16S gene sequence fragments of Bovine to optimize the amplification temperature and time.The rapid detection method was compared with the commercial fluorescent quantitative PCR kit by combining the color of nucleic acid test strip.Results The optimal reaction condition of LAMP detection method was set at 65℃for 30 min,and the results could be determined by naked eye through the color of amplified products within 5 min.This method's highly specific and only beef samples showed specific amplification.The minimum detection limit is 10μg.Compared with the fluorescence PCR method,the results were consistent.Conclusion Loop-mediated isothermal amplification binding nucleic acid test strip detection method has the advantages of convenient nucleic acid extraction,strong specificity,high sensitivity,short detection time and can prevent pollution,and it is very suitable for the field rapid testing in grassroots inspection institutions,farmers'markets and supermarkets.
作者
吴晓芳
沈月华
徐德顺
卢忠豪
WU Xiao-fang;SHEN Yue-hua;XU De-shun;LU Zhong-hao(Huzhou Municipal Center for Disease Control and Prevention,Huzhou,Zhejiang 313000,China)
出处
《中国卫生检验杂志》
CAS
2021年第17期2073-2075,共3页
Chinese Journal of Health Laboratory Technology
基金
湖州市科技计划项目(2018GY23)。
关键词
环介导等温扩增
核酸试纸条
牛肉源性成分
快速检测
Loop-mediated isothermal amplification
Nucleic acid test strip
Beef derived component
Rapid detection
