A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, salmonella typhimurium, and Listeria monocytogenes pathogen bacteria
A rapid multiplexed chemiluminescent immunoassay for the detection of Escherichia coli O157:H7, Yersinia enterocolitica, salmonella typhimurium, and Listeria monocytogenes pathogen bacteria
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DOI：
10.1021/jf063600b
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发表时间：
2007-06-27
影响因子：
6.1
通讯作者：
Roda, Aldo
中科院分区：
文献类型：
--
作者：
Magliulo, Maria;Simoni, Patrizia;Roda, Aldo
建立了一种简便、快速的多重夹心荧光酶免疫分析法，用于同时检测大肠杆菌O 157：H7、小肠结肠炎耶尔森菌、鼠伤寒沙门氏菌和单核细胞增生李斯特菌。为了实现四种病原体的多重检测，设计了一种新的聚苯乙烯96孔微量滴定板格式，其中每个主孔底部包含四个子孔。将对每种细菌特异性的单克隆抗体分别固定在每个子孔中。当将样品加入到主威尔斯孔中时，能够特异性结合相应单克隆抗体的细菌被捕获在四个子威尔斯孔中的一个中。随后，加入过氧化物酶标记的抗四种细菌的多克隆抗体的混合物，并使用低光电荷耦合成像装置通过增强的基于鲁米诺的荧光混合物测量每个孔中结合的多克隆标记抗体的过氧化物酶活性。该方法简单、快速，所有菌种的定量限为10（4）-10（5）CFU/mL。通过与常规培养方法的结果比较来评价该方法的准确性，结果令人满意，回收率范围为90%至120%。该方法可用作筛选试验，以评估这些病原菌在不同食品中的存在。
A simple and rapid multiplexed sandwich chemiluminescent enzyme immunoassay has been developed for the simultaneous detection of Escherichia coli O157:H7, Yersinia enterocolitica, Salmonella typhimurium, and Listeria monocytogenes. To achieve the multiplexed detection of the four pathogens, a new polystyrene 96 well microtiter plate format has been designed, in which each main well contains four subwells in the bottom. The monoclonal antibodies specific for each bacteria were separately immobilized in each subwell. When the samples were added to the main wells, the bacteria able to specifically bind to the corresponding monoclonal antibody were captured in one of the four subwells. Subsequently, a mixture of peroxidase-labeled polyclonal antibodies against the four bacteria was added and the peroxidase activity of the bound polyclonal labeled antibodies in each well was measured by an enhanced luminol-based chemiluminescent cocktail using a low-light charge-coupled imaging device. The assay was simple and fast, and the limit of quantification was in the order of 10(4)-10(5) CFU/mL for all bacterial species. The accuracy of the method, evaluated by comparison of the results with a conventional culturing methodology, was satisfactory, with recovery values ranging from 90 to 120%. This method can be used as a screening test to evaluate the presence of these pathogen bacteria in different foodstuffs.