Exonuclease III-assisted recycling amplification detection of hepatitis B virus DNA by DNA-scaffolded silver nanoclusters probe

Abstract

An exonuclease III-assisted recycling amplification (ERA) coupled with fluorescent DNA-scaffolded silver nanoclusters (Ag NCs) for sensitive determination of hepatitis B virus (HBV) DNA is proposed. The sensing mechanism is based on the employment of two different molecular beacons (MBs), MB1 and MB2. In the absence of targets, the MBs self-hybridize into stable stem-loop structures with exonuclease III (Exo III) cleaving resistance, and the MB2 containing a C-rich loop can be utilized to synthesize Ag NCs with high fluorescence signal. In the presence of targets, the targets can bind with MB1 to form partly duplex structure. With the cleaving of Exo III, the liberated target DNA can initiate the cycle of MB1-target hybridization, while the generated trigger DNA can hybridize with MB2, inducing a conformational change of MB2 from hairpin to single-stranded structure. The structural change of MB2 would influence the formation of Ag NCs, thus producing weak fluorescence signal, and the released trigger DNA could activate the cycle of MB2-trigger hybridization. The present method for HBV DNA analysis exhibits a detection limit of 0.97nM (S/N=3) with high specificity, and demonstrates its applicability for the HBV detection in human serum.