Magnetic poly(phages) encoded probes-based dual-mode assay for rapid determination of live Escherichia coli and Hafnia paralvei based on microfluidic chip and ATP bioluminescence meter.

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.