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重组酶等温扩增试纸条快速检测阪崎克罗诺杆菌

陈纯阳1,张宸宁1,史爱莹1,杜欣军1,王硕2   

  1. 1. 天津科技大学食品工程与生物技术学院
    2. 天津科技大学
  • 收稿日期:2019-04-12 修回日期:2019-10-10 出版日期:2019-12-25 发布日期:2019-12-24
  • 通讯作者: 杜欣军 E-mail:xinjundu@163.com
  • 基金资助:
    国家重点研发计划项目

Rapid Detection of Cronobacter sakazakii using Recombinase Polymerase Amplification Combined with Lateral Flow Strip

  • Received:2019-04-12 Revised:2019-10-10 Online:2019-12-25 Published:2019-12-24

摘要: 阪崎克罗诺杆菌是一种食源性条件致病菌,它会导致人类不同年龄人群、特别是新生儿和免疫功能不健全的婴幼儿发生严重疾病。本研究将重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)与免疫层析试纸条(lateral flow strip,LF)相结合,建立了一种阪崎克罗诺杆菌快速检测方法(RPA-LF)。本研究将引物分别用生物素和地高辛修饰后进行RPA扩增,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。

关键词: 重组酶聚合酶等温扩增技术, 免疫层析试纸条, 阪崎克罗诺杆菌, 快速检测

Abstract: Cronobacter sakazakii is an opportunistic food-borne pathogen that causes serious diseases in humans of different age groups, especially newborns and immunocompromised infants. In this study, recombinase polymerase isothermal amplification (RPA) cobined with lateral flow test strips (LF) was developed to quickly and reliably detect C. sakazakii. RPA amplification using the primers modified with biotin and digoxin produced large amount 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, and finally showed visible results on the strip. RPA-LF method showed detection limit of 1.7×102 CFU/mL for C. sakazakii in pure culture and no cross-reactivity with other common pathogens. Performance of the RPA-LF for atrifically contaminated food samples was evaluated and the results showed that the detection limit was 1.7×100 CFU/g after 4 h or 6 h enrichment for different food types. In order to further evaluate the performance of the method developed in this study, 20 real food samples were detected by the RPA-LF and the national standard method simultaneously. The results showed that test results of the RPA-LF were in agreement with that of national standard method of China.

Key words: recombinase polymerase isothermal amplification (RPA), lateral flow test strips (LF), Cronobacter sakazakii, rapid detection

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