Lysin cell-binding domain-functionalized magnetic beads for detection of Staphylococcus aureus via inhibition of fluorescence of Amplex Red/hydrogen peroxide assay by intracellular catalase
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Accurate and rapid identification of Staphylococcus aureus (S. aureus) is of great significance for controlling the food poisoning and infectious diseases caused by S. aureus. In this study, a novel strategy that combines lysin cell-binding domain (CBD)-based magnetic separation with fluorescence detection was developed for the specific and sensitive quantification of S. aureus in authentic samples. The S. aureus cells were separated from the sample matrix by lysin CBD-functionalized magnetic beads. Following lysis by lysostaphin, intracellular catalase was released from S. aureus cells and detected by a fluorometric system composed of horseradish peroxidase (HRP), hydrogen peroxide (H2O2), and Amplex Red. S. aureus was quantified via the inhibitory effect of the released intracellular catalase on the fluorometric system since the catalase could decompose the H2O2. Optimized conditions afforded a calibration curve for S. aureus ranging from 1.0 × 102 to 1.0 × 107 CFU mL−1. The detection limit was as low as 78 CFU mL−1 in phosphate-buffered saline (PBS), and the total detection process could be completed in less than 50 min. Other bacteria associated with common food-borne and nosocomial infections negligibly interfered with S. aureus detection, except for Staphylococcus epidermidis, which may have slightly interfered. Moreover, the potential of this proposed method for practical applications has been demonstrated by detection assays of sterilized milk and human serum.
KeywordsStaphylococcus aureus Lysin cell-binding domain Magnetic separation Catalase Amplex Red
This work was supported by the Natural Science Foundation of Shandong Province, China (Grant Nos. ZR2017LH057 and ZR2018ZC1054), and the National Natural Science Foundation of China (Grant Nos. 81770915 and 81802054).
Compliance with ethical standards
Informed consent for the human serum used in this study was obtained from the individual participants according to the World Medical Association Declaration of Helsinki. The studies have been approved by the ethics committee of Weifang Medical University and have been performed in accordance with the ethical standards.
Conflict of interest
The authors declare that they have no conflict of interest.
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