Development of an on-spot and rapid recombinase polymerase amplification assay for Aspergillus flavus detection in grains
Development of an on-spot and rapid recombinase polymerase amplification assay for Aspergillus flavus detection in grains
复制标题
DOI：
10.1016/j.foodcont.2021.107957
复制
发表时间：
2021-02
期刊：
影响因子：
6
通讯作者：
Qijun Liu;Xu Li;Wu Riqin-;Xinglong Xiao;F. Xing
中科院分区：
文献类型：
--
作者：
Qijun Liu;Xu Li;Wu Riqin-;Xinglong Xiao;F. Xing
黄曲霉是粮食中的主要污染物之一，大多数菌株都含有黄曲霉。黄曲霉毒素对食品安全和人体健康构成极大威胁。及早发现食物受甲类污染。黄曲霉毒素能有效地降低AFs对人类健康的危害，防止AFs的进一步传播。黄的本研究建立了一种灵敏、快速的现场检测方法。已经开发了基于重组酶聚合酶扩增（RPA）的黄素颗粒。设计了3对RPA引物，筛选出一对适合的引物。建立了一种简便、高效的DNA提取方法，并应用于现场检测。提供两个温度，环境温度（AT，26 ± 1 °C）和体温（BT，36 ± 1 °C）用于检测。在AT和BT条件下分别对反应时间和引物浓度进行了优化。荧光DNA染料用于视觉检测。BT和AT分别在20 min和30 min内完成整个检测试验。敏感性试验表明，该方法的检出限为101个孢子/g。该方法的特异性与实时荧光PCR法相当。新的RPA检测方法在其他领域也有很大的应用潜力。
Aspergillus flavusis one of the main contaminants in grains, and most strains ofA. flavuscan produce aflatoxins (AFs), which poses a great threat to food safety and human health. Early detection of food contaminated byA. flavuscan effectively reduce the harm of AFs to human health and prevent further spread ofA. flavus. In this study, a sensitive and rapid method for on-spot detection ofA. flavusin grains, based on recombinase polymerase amplification (RPA), has been developed. Three RPA primer pairs were designed, and then a suitable primer pair was selected. A simplified and efficient DNA extraction method for on-spot detection was developed and applied. Two temperatures, ambient temperature (AT, 26 ± 1 °C) and body temperature (BT, 36 ± 1 °C), were provided for the detection. The reaction time and primer concentration were optimized at AT and BT, respectively. And a fluorescent DNA dye was used for the visual detection. The whole detection-assay can be completed within 20 min at BT and 30 min at AT, respectively. The sensitivity evaluation proved that the limit of detection (LOD) of the RPA-assay was 101conidia/g. The specificity of the assay was equal to the real-time PCR. The new RPA-assay also had great potential in other fields′ applications.