A Study Of Detection Methods On Staphylococcus Aureus Based On Flow Cytometry

Staphylococcus aureus is one of the most important food-borne pathogens. Food-borne diseases caused by S.aureus have attracted great attention around the world. Rapid detection of S.aureus is an effective means of prevention and control of bacterial food poisoning. Traditional detection method requires bacterial cultivation and a series of complicated biochemical tests for identification of bacteria, which is time-consuming and can’t meet the demand of the current rapid detection. Flow cytometry is a detection technology integrating optics, electrics and immunology, which could rapidly provide multi-parameter qualitative and quantitative analysis data for a single bacteria or microsphere in the liquid environment. Now this technology is widely used in the routine work of bacteria and microbiological studies: quickly counting the number of bacteria separating from water and food samples of microbial contamination, identifying target bacteria in combination with a fluorescein-stained ligand, establishing multi-detection methods after a preparation of ligand functionalized microspheres, etc. Now, the mainly ligands for bacteria detection are antibody and aptamer. Aptamer is a class of nucleic acid molecules obtained by SELEX(Systematic Evolution of Ligands by Exponential Enrichment,SELEX), which has a similar identifying mechanism with antibody, nevertheless, aptamer has a number of advantages over antibody: chemical stability, short-time screened cycle, easy to be modified and to facilitate construction of biosensors combining with other detection methods, etc. Currently, aptamer that has high-affinity and can be used directly in the practical application is difficult to be screened, therefore, we should be cautious of selecting when constructing detection biosensor based on aptamer or use antibody and aptamer in combination if necessary.We aim to establish S.aureus detection method based on ligand recognition using flow cytometry, and also make exploratory research on simultaneously detection of S.aureus and E.coli O157:H7. The detailed contents are described as follow.(1) Detection method of flow cytometry based on aptamer recognition for S.aureusIn our experiment, FAM fluorescein-labeled aptamer SA17 was used to specifically recognize S.aureus and then detected by flow cytometry which could give an accurate percentage of positive bacterial binding with fluorescent aptamer. By optimizing the aptamer folding method, the concentration of aptamers detection, we established a direct detection method of flow cytometry based on ligand recognition after a verification of specificity and sensitivity. Finally, an exploratory experiment of using two different fluorescein-labeled ligands was conducted to detect S.aureus and E.coli O157:H7 in one sample.For the convenience of the next analysis, we regulated flow cytometry FSC/SSC voltage as logarithmic mode to gate the bacteria. Firstly, S.aureus was incubated with FAM fluorescein-labeled aptamer SA17 to compare the detection efficiency of aptamers in four kinds of different folding methods. ANOVA analysis showed that detection effect of aptamers in four kinds of folding methods are significant differences. LSD analysis displayed that the detection efficiency of aptamer folded without high temperature denaturation has a significant differences compared with the other three folding methods. So we determined the working concentration of aptamer as 250 nM under the optimal folding method. The entire course of this experiment could be completed in 40 min after a pre-amplification of S.aureus to above 108 CFU. Finally, we used PE-streptavidin-biotin-labeled E.coli O157:H7 monoclonal antibody and FAM fluorescein-labeled SA17 to detect the suspension of the two kinds of bacteria, the identification result of which was compared with the detection efficiency of only sigle ligand against the corresponding bacteria, due to SA17 aptamer having no cross-linking with E.coli O157:H7, the results showed that SA17 can specifically identify S.aureus from the mixed bacterial suspension.(2) Detection method of liquid-chip based on ligand recognition for S.aureusFirstly, S.aureus polyclonal antibody and aptamer functionalized magnetic microsphere were prepared in order to enrich the target bacteria by magnetic separation, then we used fluorescein-stained aptamer and polyclonal antibody as detection ligand respectively. FCM was used to analyse fluorescent signal of detection ligand and subsequently compared the detection results of liquid chip of four patterns. Finally, we determined the suspension array technology based on double-polyclonal antibody-sandwich identification for the next study, and optimized working concentration of the detection antibody and verified the specificity and sensitivity of this method.We determine it as the positive result when the ratio of experimental group MFI and control group MFI is greater than 3. By comparing the four patterns of liquid-chip, we found that only when the capture ligand and detection ligand both are S.aureus polyclonal antibody, liquid-chip’s result can meet the criteria. Then the working concentration of detection antibody labeled with biotin was optimized as 12 μg/m L and the detection method was verified in aspect of specificity and sensitivity. The specificity results showed that the established detection method of S.aureus has no cross-linking reaction with other five common food-borne pathogens(Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritidis, E.coli O157:H7, Enterobacter sakazakii), and the sensitivity results showed this method has a detection limit of 106 CFU. At the same time, E.coli O157:H7 monoclonal antibody functionalized carboxy microsphere MEC-26 was also prepared according to the method described above, and a liquid-chip detection method for E.coli O157:H7 was established which subsequently proved to be having no cross-linking reaction with S.aureus. Finally, we used the mixture of MEC-26 and PSA-65 to detect food-borne pathogens, the results showed that both the two function microspheres cann’t identify the other four food-borne pathogens(Listeria monocytogenes, Salmonella typhimurium, Salmonella enteritidis, Enterobacter sakazakii). And PSA-65 is capable of specifically recognizing S.aureus from the mixed bacterial suspension of S.aureus and E.coli O157:H7.In this study, we established a direct detection method of flow cytometry based on specific ligand recognition and indirect detection method of liquid-chip based on capture beads. Using aptamer FAM-SA17 to identify S.aureus without folding had avoid the time consuming on pre-treatment process of aptamer, which make the detection process more time-saving and convenient. In the indirect liquid-chip detection methods, we prepared antibody functionalized magnetic microspheres to separate target bacteria from the sample before FCM analyzing the intensity of fluorescent reporter molecule, making the results more real and reliable. Both the two methods provide new ideas for the establishment of(multiplex) detection methods which is promising in the area of food safety.