摘要
将重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)与免疫层析试纸条(lateral flowstrip,LF)相结合,建立一种阪崎克罗诺杆菌快速检测方法(RPA-LF)。将引物分别用生物素和地高辛修饰后进行RPA,产生生物素和地高辛标记的双链DNA产物,扩增产物与胶体金标记的地高辛抗体及试纸条上固定的链霉亲和素发生特异性结合,最终在试纸条上呈现肉眼可见的结果。灵敏度分析结果显示,该方法对纯培养目标菌的检测限为1.7×102 CFU/mL,特异性分析结果显示RPA-LF方法与其他常见病原菌无交叉反应。利用人工污染的食物样品评估RPA-LF检测效果,结果显示不同样品增菌4 h或6 h后,检测限达到1.7×100 CFU/g。利用20 种实际样品作为检测对象、利用国标方法作为对照,评估方法的检测效果,结果显示本研究所建立的方法与国家标准检测方法获得一致的检测结果。
In this study, recombinase polymerase isothermal ampli fication (RPA) combined with lateral flow test strips (LF) was developed to quickly and reliably detect Cronobacter sakazakii. RPA using the primers modi fied with biotin and digoxin produced large amounts of double-stranded DNA products labeled with biotin and digoxin at each end. The products could interact with gold-labeled anti-digoxin antibody and streptavidin fixed on the LF, giving visible results on the strip. The RPA-LF method showed a limit of detection (LOD) of 1.7 × 102 CFU/mL for C. sakazakii in pure culture with no cross-reactivity with other common pathogens. The LOD of the RPA-LF was 1.7 × 100 CFU/g after 4 or 6 h enrichment for different types of atrifically contaminated food samples. In order to further evaluate its performance, 20 real food samples were detected by the RPA-LF in comparison with the national standard method. The results obtained were consistent with each other.
作者
陈纯阳
张宸宁
史爱莹
杜欣军
王硕
CHEN Chunyang;ZHANG Chenning;SHI Aiying;DU Xinjun;WANG Shuo(State Key Laboratory of Food Nutrition and Safety,College of Food Science and Engineering,Tianjin University of Science and Technology,Tianjin 300457,China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2019年第24期306-312,共7页
Food Science
基金
“十三五”国家重点研发计划重点专项（2018YFC1603800）
