Chemiluminescence Protocols For The Detection Of Pathogens Based On Antibiotic-affinity Strategy

Pathogen is a kind of microorganism which can infect the host through air, food, water and biological contact. Staphylococcus aureus(S. aureus) is a widely distributed Gram-positive bacterium. It has been one of the major foodborne and iatrogenic pathogens involved in a variety of dangerous diseases including septicemia, osteomyelitis, pneumonia, toxic shock syndrome, and endocarditis. Streptococcus mutans(S. mutans) is a facultatively anaerobic, gram-positive coccus commonly found in the human oral cavity which contributes significantly to tooth decay. Thus, it is of great importance to develop rapid, sensitive, specific and low-cost methods for S. aureus and S. mutans detections.Chemiluminescence is light emission resulted from chemical reactions. Chemiluminescence analysis is a molecular luminescence spectra analysis method. It is mainly on the basis of the linearity between the concentration of analytes and chemical luminescence intensity. In this study, two facile CL methods were developed for dual-recognition detection of pathogens by using functionalized-magnetic beads(MBs).(1) A facile CL method using antibiotic-functionalized MBS for S. aureus detection.In this work, vancomycin was used as one of the molecular recognition agent for S. aureus detection due to its strong antibacterial effect to gram-positive bacteria. MBs were functionalized with vancomycin to concentrate and separate S. aureus effectively. Further more, rabbit immunoglobulin G tagged with alkaline phosphatase(ALP-rabbit Ig G) was used as the second recognition agent and produce the chemiluminescence. The three reactants was mixed with one-step protocol, thus the detection time could be within 75 min. The linear range for S. aureus detection was 12-1.2 × 106 CFU m L-1, with a very low detection limit of 3.3 CFU m L-1. The whole detection process could be completed in 75 min. Six interference bacteria showed negligible interference in this study. This method was successfully used to quantitate S. aureus in lake water, milk, human urine and human saliva with acceptable recoveries ranging from 70.0% to 116.7%.(2) A novel CL method based on dual-recognition mode for S. mutans detection.A dual-recognition method for S. mutans detection was developed based on the fact that protein G in the cell wall of S. mutans can bind with rat Ig G2 a. MBs-rat Ig G2 a was adopted as the first molecular recognition agent to concentrate S. mutans. In order to improve the specificity, the ALP-vancomycin was used as the second recognition agent to identify S. mutans. The MBs-concentrated complex of rat Ig G2a/S. mutans/ ALP-vancomycin was formed after incubation and detected by microplate reader. The linear range for S. aureus detection was 24-2.4 × 105 CFU m L-1, with a very low detection limit of 8.0 CFU m L-1. This method was successfully used to quantitate S. mutans in human oral with acceptable recoveries ranging from 70.8% to 108.3%.