Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce
Large-volume immunomagnetic separation combined with multiplex PCR assay for simultaneous detection of Listeria monocytogenes and Listeria ivanovii in lettuce
复制标题
大体积免疫磁珠分离结合多重PCR检测同时检测生菜中单增李斯特菌和伊氏李斯特菌
DOI：
10.1016/j.foodcont.2015.06.048
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发表时间：
2016-01-01
期刊：
影响因子：
6
通讯作者：
Xiong, Yonghua
中科院分区：
文献类型：
--
作者：
Mao, Yan;Huang, Xiaolin;Xiong, Yonghua
多重聚合酶链反应（mPCR）已广泛应用于蔬菜中多种目标细菌的同时检测。然而，富集期对于提高mPCR方法的灵敏度是必要的。本文首次报道了大体积（10 mL）免疫磁性分离（IMS）结合mPCR快速检测生菜中单核细胞增生李斯特菌和伊氏李斯特菌的方法。系统研究了磁性纳米球表面链霉亲和素和生物素化抗李斯特菌单克隆抗体的量、免疫磁珠的量、免疫反应时间和磁分离时间等因素对免疫磁珠捕获效率的影响。对引物浓度、PCR反应条件和基因组DNA提取方法进行了优化。在最佳条件下，L。单核细胞增多症和L.在纯培养中，当目标菌浓度小于106 CFU/mL时，ivanovii的转化率大于90%;在生菜样品中，当目标菌浓度小于105 CFU/mL时，ivanovii的转化率大于80%。IMS结合mPCR检测纯培养物的最低检测限可达1.0 CFU/mL，生菜的最低检测限可达10 CFU/g。整个检测时间（包括样品制备、大体积IMS和mPCR检测）不到7小时。综上所述，所开发的基于IMS的大容量mPCR系统在食源性致病菌的常规筛查检测中具有很大的潜力，用于安全监测。(C)2015爱思唯尔有限公司版权所有。
Multiplex polymerase chain reaction (mPCR) has been widely used for the simultaneous detection of various target bacteria in vegetables. However, an enrichment period is necessary to improve the sensitivity of the mPCR method. In this paper, large-volume (10 mL) immunomagnetic separation (IMS) combined with mPCR for the rapid detection of Listeria monocytogenes and Listeria ivanovii in lettuce without further enrichment process is reported for the first time. Various parameters that affected the capture efficiency (CE) of IMS, including the amounts of streptavidin and biotinylated anti-Listeria monoclonal antibodies on the surface of magnetic nanobeads, the amount of immunomagnetic beads, immunoreaction time, and magnetic separation time, were systematically investigated. Moreover, the concentrations of primers, PCR conditions, and genomic DNA isolation for mPCR assay were optimized. Under optimum conditions, the CE of large-volume IMS for L. monocytogenes and L. ivanovii was greater than 90% when the concentration of target bacteria was less than 106 CFU/mL in pure culture, and was more than 80% when the concentration was below 105 CFU/mL in lettuce samples. The limit of detection of IMS combined with mPCR assay reached as low as 1.0 CFU/mL in pure culture and 10 CFU/g in lettuce. The overall assay time, including sample preparation, large-volume IMS, and mPCR assay, took less than 7 h. In summary, the developed large-volume IMS-based mPCR system exhibits great potential for routine screening detection of foodborne pathogenic bacteria for safety monitoring. (C) 2015 Elsevier Ltd. All rights reserved.