A fluorescence quenching-recovery sensor based on RCA for the specific analysis of Fusobacterium nucleatum. nucleatum.

Abstract

Fusobacterium nucleatum. nucleatum (Fn.n) is associated with colorectal carcinoma. A highly sensitive fluorescence quenching-recovery detection platform based on rolling circle amplification and hairpin molecular beacon technology for the specific analysis of Fn.n, hairpin MB being used to achieve double quenching. First, a specific recognition sequence target in the padlock probe was designed based on the nusG specific gene of Fn.n. The padlock probe then formed circular DNA using the ligase. After digestion, linear amplification was achieved by the phi29 DNA polymerase and the RCA primer, and a large amount of amplified products was obtained. Subsequently, the amplification products hybridized with the signal probe, leading to the opening of the hairpin structure in MBs. As a result, FAM at 5' end and BHQ-1 at 3' end became separated, which allowed for the recovery of the fluorescence signal. The proposed method showed high sensitivity and specificity for the detection of Fn.n genomic DNA (LOD as low as 0.7ngL-1) and performed well in the identification of this bacteria in real samples.