In this work, we present a magnetic bead–based immunosensorss for the determination of Escherichia coli O26 in milk. The assay uses commercial Dynabeads® for the capture of the target bacteria from 6-h enrichment broth and horseradish peroxidase–labelled polyclonal antibodies and detection of the peroxidase activity by chronoamperometry. Bovine seroalbumin was used as blocking reagent for reducing the binding of non-specific antibodies to the beads and/or other bacteria. The effectiveness of the enzyme-linked immunomagnetic electrochemical (ELIME) assay to detect low number of E. coli O26 in milk was demonstrated using spiked milk samples from five different lots with concentrations of 9.00–18.80 or 0.90–1.88 CFU in 25 mL. Three repetitions for each concentration were analysed in parallel also by real-time PCR following the standard method ISO/TS 13136:2012, using 24-h enriched cultures. Results demonstrated that the ELIME assay can reduce the time for analyses to one working day and produce reliable results that can be confirmed by overnight cultures of the beads-bacteria complexes allowing further characterization of the virulence, including the presence of Shiga toxin–encoding genes, which is important to discriminate the enterohaemorrhagic strains.

在这项工作中，我们提出了一种基于磁珠的免疫传感器，用于测定牛奶中的大肠杆菌O26。该测定法使用商业Dynabeads®从6 h富集肉汤和辣根过氧化物酶标记的多克隆抗体中捕获目标细菌，并通过计时电流分析法检测过氧化物酶活性。牛血清白蛋白被用作封闭剂，以减少非特异性抗体与磁珠和/或其他细菌的结合。酶联免疫磁电化学（ELIME）测定法检测少量大肠杆菌的有效性使用来自五个不同批次的加标牛奶样品在25 mL中的浓度为9.00-18.80或0.90-1.88 CFU的牛奶中证明了O26。遵循标准方法ISO / TS 13136：2012，使用24小时富集培养，通过实时PCR平行分析每种浓度的三个重复。结果表明，ELIME分析可将分析时间缩短至一个工作日，并可产生可靠的结果，这一点可通过隔夜培养珠-细菌复合物来证实，从而可进一步鉴定毒力，包括存在志贺毒素编码基因，这对于区分肠出血性菌株很重要。