Functional chimera aptamer and molecular beacon based fluorescent detection of Staphylococcus aureus with strand displacement-target recycling amplification

Abstract

A fluorescent detection of Staphylococcus aureus (S. aureus) is established based on a finely designed functional chimera sequence, a molecular beacon (MB), and strand displacement target recycling. The chimera sequence, which consists of the aptamer sequence of S. aureus and the complementary sequence of MB, can form a hairpin structure due to the existence of intramolecular complementary regions. When S. aureus is present, it binds to the aptamer region of the chimera, opens the hairpin and unlocks the complementary sequence of MB. Subsequently, the MB is opened and intensive fluorescence signal is restored. To increase the sensitivity of the detection, signal amplification is achieved through strand displacement-based target recycling. With the catalysis of Nb. Bpu10I nicking endonuclease and Bsm DNA polymerase, the MB sequence can be cleaved and then elongated to form a complete duplex with the chimera, during which S. aureus is displaced from the chimera and proceeded to the next round of the reaction. This assay displays a linear correlation between the fluorescence intensity and the logarithm of the concentration of S. aureus within a broad concentration range from 80 CFU/mL to 8 × 106 CFU/mL. The detection limit of 39 CFU/mL can be derived. The assay was applied to detect S. aureus in different water samples, and satisfactory recovery and repeatability were achieved. Hence the designed chimera sequence and established assay have potential application in environmental pollution monitoring.