Enzymatic repairing amplification-based versatile signal-on fluorescence sensing platform for detecting pathogenic bacteria

Abstract

A novel fluorescence biosensing strategy for ultrasensitive and specific detection of pathogenic bacteria based on target-triggered enzymatic repairing amplification (ERA) has been developed. This strategy relies on target–aptamer binding mediated ERA reaction, which is carried out cyclically with the help of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) to produce amplified fluorescence signal. In our assay, the specially designed hairpin probe (HAP) is used as DNA template responsible for producing a great quantity of reporter oligonucleotides and secondary primers, which can initiate a new cycle of polymerization-repairing amplification. Moreover, by the combination of polymerase-catalyzed incorporation of lesion bases with UDG and Endo IV-assisted ERA, multiple cycle of amplification of the recognition event is achieved, enabling ultrasensitive detection of pathogenic bacteria. Under optimal conditions, this biosensor exhibits ultrasensitivity toward target pathogenic bacteria with detection limits of 9.86cfumL−1 and a detection range of 5 orders of magnitude. Additionally, the biosensor has the ability of combating nonspecific background. Furthermore, an archer probe containing the anti-target aptamer sequence and a primer sequence is designed, which translates the binding of target to aptamer into the presence of primer sequence, enabling the detection of various targets, such as protein, DNA, small molecular, and any substance possessing its aptamer. Hence, the proposed target-triggered ERA-based signal-on fluorescence sensing strategy indeed create a versatile and useful platform for detection of pathogenic bacteria, related food safety analysis and clinical diagnosis.