Sensitive quantitative detection of bacterial DNA based on lysozyme signal probe and Exo III-aided cycling amplification reaction

Abstract

A sensitive bacterial DNA detection method based on lysozyme signal probe and exonuclease III (Exo-III) aided cycling amplification is developed. First, the capture DNA conjugated with lysozyme is assembled on magnetic beads (MBs). In the presence of a perfectly matched target bacterial DNA, blunt 3′-terminus of the capture probe is formed, thereby activating Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing lysozyme and target DNA. The released target DNA then finds another capture probe to start a new cycle, while the released lysozyme is recovered by magnetic separation for fluorescence signal collection. Because of the separating function of MBs, unique cleavage function of Exo-III, and catalytic signal amplification of lysozyme, this system achieves sensitive bacterial DNA detection, with a detection limit of 3.5fM. Besides this sensitivity, this strategy exhibits excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets, as well as good applicability in real human serum samples. As a result, this strategy shows considerable potential for qualitative and quantitative analysis of bacteria.