Lysozyme as sensitive reporter for fluorometric and PCR based detection of E. coli and S. aureus using magnetic microbeads

Abstract

We describe the use of lysozyme as a sensitive reporter for detection of bacteria. The assay comprises the following steps: First, specific primers are used for targeting the rDNA of E. coli and S. aureus, respectively, and the polymerase chain reaction is applied to amplify bacterial DNA. Then, the target DNA binds to the capture probe immobilized on magnetic beads, with lysozyme acting as signal reporter that catalyzes the hydrolysis of an enzyme substrate to form the blue fluorescent product 4-methylumbelliferone. Coupled with multiple lysozyme substrates, this fluorometric detecting method has a detection limit of 50 CFUa <...mL(-1) bacteria. The optical changes may also be detected by colorimetry and monitored with bare eyes. The method was selective over other bacteria and was successfully applied to the determination of E. coli and S. aureus in human serum, which provides a perspective for lysozyme application and species-specific pathogenic bacteria detection.