摘要
市场中肉类食品掺假的复杂多样化使得监管机构对动物源性成分鉴定的便捷性、准确性、灵敏性要求越来越高。尤其是市场上经济价值较高的牛肉类产品已成为制假的重灾区,迫切需要建立一种快速、高效的牛源性成分分子检测方法。基于此,以牛组成型表达基因β-actin为靶基因设计并筛选了几组牛特异性扩增的引物和探针组合,经过灵敏度检测和特异性验证,建立了一种牛源性成分实时荧光重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)快速检测方法。通过优化试剂配比,提高了一步法提取牛肉制品DNA的效率,同时将PRA技术与胶体金免疫试纸条显色技术相结合实现了检测的便捷化和结果的可视化。实际应用结果表明,研究建立的牛源性RPA检测方法能够特异性地检测牛源性成分,且最低检测灵敏度可达到14.8个拷贝,通过胶体金免疫试纸条得到的可视化结果的准确性和灵敏度与实时荧光RPA相同。该方法特异性强、灵敏度高,整个过程在25 min内即可完成,极大缩短了检测时间。
The diversification of meat adulteration in the market has made it increasingly difficult for regulatory agencies to identi‐fy ingredients of animal origin with ease,accuracy and sensitivity.In particular,beef products with high economic value on the market have become the hardest hit areas for counterfeiting.Therefore,it is urgent to establish a fast and efficient molecular de‐tection method for bovine components.Based on the requirement,this study designed and screened several groups of bovine spe‐cific amplified primers and probe combinations withβ-actin,a reference gene expressed constitutively in cattle.Through sensitivity detection and specificity verification,recombinase polymerase amplification(RPA)method for rapid detection of bovine-derived components was established.At the same time,the efficiency of one-step DNA extraction from beef products was improved by adjusting and optimizing the reagent ratio.PRA technology was combined with colloid gold immunotest strip color develop‐ment technology to realize the convenience of detection and visualization of results.Application results showed that the proposed method could detect bovine RPA specifically,with a minimum detection sensitivity of 14.8 copies,and could visualize the visual‐ized results obtained by colloidal gold immunotest strips,and the accuracy and sensitivity of the results were the same as that of real-time fluorescence RPA.The method has strong specificity and high sensitivity,and the whole process can be completed in 25 min,greatly shortening the detection time.
作者
王晓雪
纪艺
余卉茹
徐俊锋
彭城
汪小福
李玥莹
陈笑芸
WANG Xiaoxue;JI Yi;YU Huiru;XU Junfeng;PENG Cheng;WANG Xiaofu;LI Yueying;CHEN Xiaoyun(College of Life Sciences,Shenyang Normal University,Shenyang 110000,China;State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products,Key Laboratory of Traceability for Agricultural Genetically Modified Organisms,Ministry of Agriculture and Rural Affairs,Institute of Quality,Safety and Nutrition of Agricultural Products Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Xianghu Laboratory,Hangzhou 311258,China)
出处
《生物技术进展》
2024年第2期278-286,共9页
Current Biotechnology
基金
科技创新2030重大项目农业生物育种重大专项(2022ZD040212)
浙江省重点研发项目(2021C02059)。
关键词
牛源性成分
重组酶聚合酶扩增技术
胶体金免疫试纸条
bovine-derived components
recombinase polymerase amplification
colloidal gold immunoassay strips
