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Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium that causes a wide range of illnesses, necessitating the development of new technologies for its detection. Herein, we propose a graphene oxide (GO)-based sensing platform for the detection of mecA gene in MRSA using flap endonuclease 1 (FEN1)-assisted target recycling and Klenow fragment (KF)-triggered signal amplification. Without the target, all the DNA probes were adsorbed onto GO, resulting in fluorescence quenching of the dye. Upon the addition of the target, a triple complex was formed that triggered FEN1-assisted target recycling and initiated two polymerization reactions with the assistance of KF polymerase, generating numerous dsDNA that were repelled by GO. These dsDNAs triggered fluorescence enhancement when SYBR Green I was added. Therefore, the target DNA was quantified by measuring the fluorescence at excitation and emission wavelengths of 480/526 nm. This mecA gene assay showed a good linear range from 1 to 50 nM with a lower limit of detection of 0.26 nM, and displayed good applicability to the analysis of real samples. Thus, a new method for monitoring MRSA has been developed that has great potential for early clinical diagnosis and treatment. • A novel GO-based bioassay was constructed for efficient assay of mecA gene in MRSA. • Integration of FEN1 and KF in one system that trigger signal amplification to improve the bioassay performance. • The bioassay can achieve low detection limits of 0.26 nM within 5 min. • The bioassay was successfully applied to determination of MRSA in real samples. [ABSTRACT FROM AUTHOR]

We describe a graphene oxide (GO)-based bioassay for the fluorometric determination of norA gene transcription (mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This approach is based on Nb.BbvCI-assisted target recycling (NATR) and T7 exonuclease (T7 Exo)-triggered cascade dual-recycling signal amplification (TTCDRSA). The system included GO, a capture probe (CP), an assistant probe (AP), two carboxyfluorescein (FAM)-labeled hairpins (HP1 and HP2), endonuclease Nb.BbvcI, and exonuclease T7. In the presence of a target, AP, together with the target RNA, can hybridise with CP via partial complementarity to one another and open its hairpin structure to form a triple complex that is recognised by Nb.BbvCI. Once the CP is cleaved, the released AP and target RNA can walk on the carboxylated graphene oxide (CGO) surface to bind with another CP which induces the next round of cleavage, accumulating many trigger probes (TPs). The TPs then activate TTCDRSA with the assistance of T7 Exo, HP1, and HP2 to produce large amounts of free FAMs. These free FAMs are repelled by GO and exhibit enhanced fluorescence signals at excitation/emission wavelengths of 480/514 nm. The limit of detection (LOD) of the bioassay was calculated to be 0.37 fM, and the linear range of the method ranged from 1 fM to 1 nM. More importantly, the bioassay also exhibited high sensitivity and selectivity for target RNA detection in real samples, which may open a new promising avenue for monitoring drug efflux and studying the mechanisms of drug actions. (a) NorA efflux pump regulated by ArlS/ArlR two-component regulatory system and baicalin suppresses the NorA efflux pump formation in MRSA; (b) Schematic diagram showing the principle of the graphene oxide-based fluorescent bioassay for norA gene transcription based on Nb.BbvCI-assisted target recycling and T7 exonuclease-triggered cascade dual recycling signal amplification. [Display omitted] • An ultrasensitive GO-based assay for detecting norA gene transcription in MRSA was developed. • The integration of target cycling and signal amplification greatly improved the sensitivity of the assay. • A detection limit of 0.37 fM was achieved within 40 min. • The assay was successfully applied to monitor drug efflux and reveal the mechanism of during action. [ABSTRACT FROM AUTHOR]