Chemiluminescent Protocols For The Detection Of Gram-positive Bacteria Utilzing The Affinity Of Glycopeptide Antibiotics

Gram-positive bacteria are a group of pathogenic bacteria that are mainly harmful to human and animals.For example,Staphylococcus aureus(S.aureus)is believed to be one of the leading causes of alimentary toxicosis,septicemia and sepsis.Bacillus cereus and Micrococcus luteus can also cause alimentary toxicosis and infectious diseases.Streptococcus mutans(S.mutans)is frequently isolated from the human oral cavity,and is the prime causative organisms of human dental caries.Due to the threat of Gram-positive bacteria to human and animals,it is important to develop rapid,sensitive,specific and low-cost strategies for Gram-positive bacteria detection.On the basis of the linearity between the concentration of samples and the chemiluminescence(CL)intensity of the chemical reaction system,CL analysis is a trace analysis method which determines the concentration of samples according to the CL intensity of the chemical reaction system.Bioluminescence(BL)is a kind of CL that occurs in biological systems.It can emit visible light during the process of chemical reaction.Adenosine triphosphate(ATP)-BL is one of the most common BL.It can produce highly intensive light emission when ATP reacts with luciferase,luciferin and oxygen.In this work,two facile CL protocols were developed for the detection of Gram-positive bacteria utilizing the affinity of glycopeptide antibiotic with the bacterial cell wall.(1)A facile bioluminescent protocol using vancomycin-functionalized magnetic particles for detection of the total amount of viable Gram-positive bacteria.In this study,a facile bioluminescent protocol was developed to detect the total amount of viable Gram-positive bacteria based on a novel antibiotic-affinity strategy.Vancomycin-functionalized magnetic particles were adopted as a novel molecular recognition agent to isolate and concentrate Gram-positive bacteria from sample matrix.Then the BL signal from the bacterial intracellular ATP was collected to quantify the captured bacteria after cells lysis.The developed protocol was applied to detect four Gram-positive bacteria,including Staphylococcus aureus,Micrococcus luteus,Bacillus cereus and Streptococcus mutans.It showed a linear range of 1.0 × 102-1.0 × 107 CFU m L-1 and a detection limit of 33 CFU m L-1 for the four model bacteria.Gram-negative bacteria and dead bacteria all showed negligible interference to the detection of the viable Gram-positive bacteria.The method was successfully applied to quantify the amount of viable Gram-positive bacteria in food and pharmaceutical samples with acceptable recovery values ranging from 72.0% to 120.0%.The protocol possessed numerous advantages such as high sensitivity,low cost,ideal specificity and short detection time.It shows great application potential in food safety control and medical diagnosis.(2)A specific CL protocol for dual-site recognition of Streptococcus mutans.In this study,a novel dual-site recognition protocol was developed for CL detection of Streptococcus mutans(S.mutans)based on a designed antibiotic-affinity strategy.Teicoplanin was adopted as a novel molecular recognition agent to functionalize magnetic particles and recognize S.mutans.To achieve ideal specificity for S.mutans detection,rat immunoglobulin G2a(rat IgG2a)tagged with horseradish peroxidase(HRP)was used as the second recognition agent and signal tracer since Fab region of rat IgG2 a could bind with streptococcal protein G highly expressed in the cell wall of S.mutans.Thus HRP-tagged sandwich complex of teicoplanin/S.mutans/rat IgG2 a was formed on the magnetic particles,followed by a CL quantification of S.mutans based on a HRP-catalyzed luminol-H2O2-p-iodophenol CL reaction.This protocol showed a linear range of 1.0 × 102-1.0 × 106 CFU mL-1 and a detection limit of 33 CFU m L-1 for S.mutans detection.The recovery tests for food,pharmaceutical and biological samples showed acceptable recovery values between 83.0% and 110.0%.This developed strategy displayed great specificity,high sensitivity and short assay time,thus provided a new way for pathogenic bacteria rapid detection in complex samples.