| Foodborne disease caused by microorganisms has been the main matter of food security.For consumers, the diseases caused by microorganisms also are the largest and most directharm. Staphylococcus aureus (SA), as one of the most common food poisoning pathogenicbacteria, widely exists in the nature, so food is apt to be polluted by it. It is necessary to take arapid, effective, simple and feasible method to detect and real time monitor the contaminationof Staphylococcus aureus in food processing, storage and marketing and other aspects, toguarantee food security and health of comsumers.In this study, the nanobeads with streptavidin and Staphylococcus aureus antibody withbiotin were used to make immunomagnetic nanobeads (IMBs), and the reaction condition ofimmunomagnetic capture was optimized by test. By the specific binding between antibody onIMBs and target bacteria in the sample, the target bacteria was separated from the sample, andusing immuno-QDs as fluorescent label, the bead-bacteria-QD compounds were obtained forfluorescent detection. The calibration model between fluorescent intensity and concentrationof SA was established to achieve the rapid and quantitative detection. The main results areshown as follows:(1) In the immunomagnetic capture, experiment results showed that order of influence onthe capture efficiency of SA was addition amount of antibody coating the magnetic beads>response time> addition amount of IMB in the scope of test, and the factors of additionamount of antibody coating the magnetic beads and response time were significant factors, thefactor of addition amount of IMB was non-significant one. The optimal conditions for capturewere the amount of antibody30μL, the amount of IMBs80μL, and response time45min,respectively.(2) The optimal conditions for capture were feasible, and the capture efficiency wasstable relatively in the range of concentration from10~3to10~6CFU/mL. This method had ahigh sensitivity and specificity. It could capture the concentration of101CFU/mL SA, andShigella flexneri did not effect on the IMBs capture of SA.(3) By labeling QDs and fluorescent detection, a linear relationship between logarithm ofStaphylococcus aureus concentration in original bacterium liquid (lg N) and the fluorescenceintensity (FI) was found for the concentration ranging from10~3to10~6CFU/mL. The regression model can be expressed as FI=7.6759log N+35.056with R~2=0.9412, meanwhilea linear relationship between logarithm of captured Staphylococcus aureus concentration (lgN’) and the fluorescence intensity (FI) was found with the regression model FI=7.6944lg N’+38.042with R~2=0.9453. By calculating the recovery, it showed this method was feasible.(4) The SA in lamb wash water sample were separated by IMBs, the results showedcapture efficiency was lower than that in PBS. The lamb wash water sample with106CFU/mLSA was detected by the method, the results showed that the fluorescent emission peak was notobvious, fluorescent intensity was low, so it needs to decrease the interference of samplematrix, to let it use for food sample detection.In this research, immunomagnetic separation technique and QD fluorescence labelingtechnique were utilized together, to establish rapid, quantitative immune fluorescencedetection method for SA. The total detection time was within2h, which can satisfy thedemand of real-time detection. The detection of SA in food sample was explored initially, andit provided for reference frame for the following study on detection in food sample. |
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