A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification

•This work presents a novel magneto-DNA duplex probe.•It is a new way of improving DNA detection sensitivity based on fixed-diameter microsphere.•Coupled with exonuclease III, a detection limit of 14 pM was obtained.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3′-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

Graphical abstractDifferent from the traditional Exo-III aided cycling amplification, a novel perspective of magneto-DNA duplex probe modified on fixed-diameter magnetic microparticle was developed. This kind of bacterial DNA detection method is rapid, selective, and sensitive with a detection limit of 14 pM.Figure optionsDownload full-size imageDownload as PowerPoint slide