Zhejiang Pharmaceutical University, Ningbo, 315100, Zhejiang, China.
Key Laboratory of Advanced Mass Spectrometry and Molecular Analysis of Zhejiang Province, Institute of Mass Spectrometry, School of Material Science and Chemical Engineering, Ningbo University, Ningbo, 315211, China.
Mikrochim Acta. 2024 Nov 27;191(12):765. doi: 10.1007/s00604-024-06809-z.
A dual-mode assay was developed for screening and detecting live Escherichia coli (E. coli) and Hafnia paralvei (H. paralvei) (as two typical pathogens in aquatic environments) based on magnetic poly(phages) encoded probes (MPEP). The probes were prepared by grafting a large number of phages targeting different target bacteria on a long-chain DNA structure, respectively. They could specifically capture and enrich E. coli and H. paralvei by magnetic separation. Then, different DNA signal tags with different lengths conjugate with the corresponding MPEP-bacteria complex and form two kinds of sandwich structures, respectively. After that, the captured E. coli and H. paralvei were lysed to release both adenosine triphosphate (ATP) and DNA signal tags. The measurement includes two steps. Firstly, a portable ATP bioluminescence meter was employed to rapidly screen the positive samples that contain either of the two target bacteria. Secondly, only positive samples were injected into the microfluidic chip which could detect various DNA signal tags for accurate quantification of the target bacteria. The assay demonstrated high sensitivity (3 CFU/mL for E. coli and 5 CFU/mL for H. paralvei), high specificity (strain identification), signal amplification (20-fold), and short time (≤ 35 min). It can be applied to detect other pathogens solely by changing the relative phage in MPEP. Furthermore, the proposed dual-mode assay provides a wide prospect for rapid screening and accurate determination of live foodborne pathogens. Clinical Trial Number: nbdxms-20240322.
基于磁聚合噬菌体编码探针（MPEP），开发了一种用于筛选和检测水生环境中两种典型病原菌——大肠杆菌（E. coli）和哈夫尼亚 paralvei（H. paralvei）的双模式检测法。探针是通过分别在长链 DNA 结构上嫁接大量针对不同目标细菌的噬菌体制备的，它们可以通过磁分离特异性捕获和富集 E. coli 和 H. paralvei。然后，不同长度的不同 DNA 信号标签与相应的 MPEP-细菌复合物结合，分别形成两种夹心结构。之后，捕获的 E. coli 和 H. paralvei 被裂解以释放三磷酸腺苷（ATP）和 DNA 信号标签。该测量包括两个步骤。首先，使用便携式 ATP 生物发光计快速筛选含有两种目标细菌之一的阳性样本。其次，只有阳性样本被注入微流控芯片，该芯片可以检测各种 DNA 信号标签，从而对目标细菌进行准确的定量。该检测法表现出高灵敏度（E. coli 为 3 CFU/mL，H. paralvei 为 5 CFU/mL）、高特异性（菌株鉴定）、信号放大（20 倍）和短时间（≤35 分钟）。通过改变 MPEP 中的相对噬菌体，可以应用于检测其他病原体。此外，该双模式检测法为快速筛选和准确确定食源性病原体提供了广阔的前景。临床试验编号：nbdxms-20240322。
