One immunomagnetic separation (IMS) assay based on immunomagnetic beads (IMBs) has been evaluated as a potential pretreatment tool for the separation and enrichment of target bacteria. In this study, we successfully immobilized antibodies onto magnetic bead surfaces to form IMBs through biotin and a streptavidin (SA) system to capture viable but nonculturable (VBNC) Cronobacter sakazakii (C. sakazakii) from dairy products. Various parameters that affected the capture efficiency (CE) of IMS, including the number of antibodies, IMBs dose, incubation time, magnetic separation time, and immunoreaction temperature, were systematically investigated. We further determined the optimal enrichment conditions for different dairy substrates to ensure maximum enrichment of target pathogens in the system. An IMS technique combining improved propidium monoazide (PMAxx) and droplet digital PCR (ddPCR) was established to detect the pathogenic VBNC C. sakazakii. The IMS-PMAxx-ddPCR method after IMBs enrichment showed higher accuracy when the VBNC C. sakazakii was under 1 Log₁₀ copies/g. The detection limit for this method in a background of powdered infant formula (PIF) was 5.6 copies/g. In summary, the developed IMS-PMAxx-ddPCR method has great potential for the analysis and detection of VBNC bacteria in food.

一种基于免疫磁珠 (IMB) 的免疫磁分离 (IMS) 分析已被评估为分离和富集目标细菌的潜在预处理工具。在这项研究中，我们成功地将抗体固定在磁珠表面上，通过生物素和链霉亲和素 (SA) 系统形成 IMB，以捕获可行但不可培养 (VBNC)阪崎肠杆菌( C. sakazakii)）来自乳制品。系统研究了影响 IMS 捕获效率 (CE) 的各种参数，包括抗体数量、IMB 剂量、孵育时间、磁分离时间和免疫反应温度。我们进一步确定了不同乳制品底物的最佳富集条件，以确保系统中目标病原体的最大富集。建立了一种结合改进的单叠氮化丙啶（PMAxx）和液滴数字PCR（ddPCR）的IMS技术来检测致病性VBNC C. sakazakii。当VBNC C. sakazakii低于1 Log ₁₀时，IMBs富集后的IMS-PMAxx-ddPCR方法显示出更高的准确性份/克 该方法在婴儿配方奶粉 (PIF) 背景下的检测限为 5.6 份/克。总之，所开发的IMS-PMAxx-ddPCR方法对于食品中VBNC细菌的分析和检测具有很大的潜力。