摘要
为消除气溶胶污染在检测中出现负面影响,实现对黄曲霉毒素产生菌的快速、可视化检测,建立一种基于热敏尿嘧啶糖基化酶(Uracil-DNA Glycocasylase,UDG酶)与横向流动试纸条(Lateral Flow Dipstick,LFD)的防污染环介导等温扩增(Loop-mediated Isothermal Amplification,LAMP)的反应体系UDG-LAMP-LFD。针对黄曲霉毒素合成关键调控基因omt-1、ver-1以及aflR设计特异性LAMP引物。当aflR、omt-1、ver-1三个基因对应的将含U碱基的扩增产物稀释10~8倍时,该体系能够达到防污染效果,且该体系针对三个目标基因的灵敏度分别达到1×10^(-3)、1×10^(-5)、1×10^(-6)ng/μL,精密度均在5%以下,具有较强特异性和抗干扰能力。建立的UDG-LAMPLFD检测体系可快速灵敏、高特异性、可视化地检测黄曲霉毒素产生菌,同时可避免气溶胶污染导致的假阳性,适用于在加工、贮藏前及时检测粮油原料中黄曲霉毒素产生菌,提前预判污染风险,实施防控,实现减损增质。
To eliminate the negative effects of aerosol contamination in the detection of aflatoxin-producing bacteria and to achieve rapid and visual detection of aflatoxin-producing bacteria,an anti-contamination loop-mediated isothermal amplification(LAMP)based on the heat-sensitive uracil-DNA glycosylase(UDG enzyme)and the lateral flow dipsticks(LFD)was developed.Rapid and visual detection of aflatoxin-producing bacteria was achieved by the UDG-LAMP-LFD system.Specific LAMP primers were designed for the key regulatory genes of aflatoxin synthesis,omt-1,ver-1,and aflR.The amplification products of aflR,omt-1,and ver-1 genes corresponding to a 108 dilution of U-base-containing amplification products were eliminated by this system,thus achieving anti-contamination effects.The sensitivity of this system for the three target genes was determined to be 1×10^(-3),1×10^(-5),and 1×10^(-6)ng/μL,respectively,and the precision was measured to be less than 5%,with strong specificity and anti-interference ability being demonstrated.Aflatoxin-producing bacteria were detected with high sensitivity,specificity,and visualisation by the UDG-LAMP-LFD assay system,while false positives caused by aerosol contamination were avoided.The UDG-LAMP-LFD system was demonstrated to be suitable for the timely detection of aflatoxin-producing bacteria in grain and oil raw materials prior to processing and storage,so that the risk of contamination was predicted in advance,prevention and control measures were implemented,and loss reduction and quality improvement were achieved.
作者
安红玉
陈晨
王艳华
卢增慧
陶泽
王可
李拖平
李苏红
AN Hongyu;CHEN Chen;WANG Yanhua;LU Zenghui;TAO Ze;WANG Ke;LI Tuoping;LI Suhong(College of Food Science,Shenyang Agricultural University,Shenyang 110866,China;Microbial Research Institute of Liaoning Province,Chaoyang 122000,China;Central Grain Reserve Xinmin Direct Warehouse Co.,Ltd.,Xinmin 110300,China;Central Grain Reserve Shenyang Direct Warehouse Co.,Ltd.,Shenyang 110000,China)
出处
《食品工业科技》
北大核心
2025年第12期324-333,共10页
Science and Technology of Food Industry
基金
沈阳市科技局社会治理科技专项(公共安全项目)(22322339)。
