| Staphylococcus aureus is a kind of environmental microorganisms which widely exist in the nature. It is a normal component of biological communities when their quantity is small. It will increase to large numbers when the environment is fit for their growth, which will spread widely through air and water, contaminate the environment and infect other organism result in causing a serious of diseases even death. It will threat the health of the other organism including human beings, and result in financial loss. S. aureus can produce enterotoxin when it infects human body through water, which will cause nausea, vomiting, stomach cramps, diarrhea and other symptoms of poisoning. The immunocompromised populations such as children and the elderly people will result in septic shock or multiple organ failure, even death. Moreover, during the process of spreading, S. aureus are likely to produce drug resistance genes, which will cause serious biosafety issues.Consequently, for the control of S. aureus contamination, the health of human beings, the prevention of biosafety issues and the early warning of pollution, it is dramatically important to detect the concentration of S. aureus. In these years, many researches about S. aureus analysis and detection have been done by the method of cultivation. As one of the biological contaminants, it can grow and reproduce in a short time. The results of concentration measurement of one sample will be different, when the detection time is different. Therefore, there is a surging need of fast, online real-time detection of S. aureus.Microfluidic technology is an emerging technology which can integrate variety of reactions into a small chip with few square centimeters. It has been one of the best pollutants detection technologies, due to its high efficiency, low costs, short analysis time, and can achieve high throughput, portable detection and complete sample to answer procedure on one chip.Two kinds of microfluidic detection system have been established in this thesis, which are microfluidic-loop-mediated isothermal amplification (LAMP) detection system and microfluidics-immunoassay systems which are capable of detecting S. aureus with the characteristics of high efficient, accurate, fast, low-cost real-time detection.In the process of microfluidic-LAMP system establishment, one set of primer has been designed and selected which can amplify the nuc gene in S. aureus. After vificated the specificity of the primer using 13 kinds of other bacteria as negative control, one macro LAMP detection system for S. aureus has been established. Then, the stable LAMP system with high specificity was integrated in microfluidic chip. This new microfluidic-LAMP system integrated sample handling, DNA release, LAMP reaction and result observation into one small chip which is centimeter level. Reducing the 25μL LAMP reaction system to 1.5μL while integrating the macro system into microchannel. Therefore, the need for regents and sample has been greatly reduced. The detection limit is 10 DNA copies which are 1-2 orders lower than PCR. The detect result can been seen clearly under 365UV lamp. And the system can be integrated into portable, automated system by optimizing the structure of the chip to easy the making and operating process. It can be used to do field detection in practical application, avoiding the trouble of sample transportation. To verify the usefulness and accuracy of the system,24 samples has been used to validated its usefulness and by comparing traditional culturing method. Using this system to real-time monitoring the concentration of S. aureus can avoid the large-scale S. aureus pollution of environment, play a positive role in S. aureus control, protect human health from the threaten of S. aureus, can be applied widely. To accommodate the need for large number of detection, the microfluidic-LAMP assay mentioned above has been optimized, result of completing 12 samples detection at one time. The making and operation process were more simple and convenient through optimizing the design of the chip. The detection cost, the need for reagents has been reduced, and the making and operation process are simple, can be integrate into portable, automated system.A microfluidic-immune system has also been established in this thesis. It can detect 8sample at one time in 40min. Its specificity has been verified. Compared to macro detection system, this system greatly reduced the detection cost and detection time. It can be used to rapid, real time, on-site S. aureus detection after optimizing the optical system. The concentration of S. aureus in sample can be detected by analyzing the fluorescence intensity, and the result can be used to do environmental analysis. To further demonstrate the feasibility and stability of the system, actual samples were used and compare with the result of traditional culture method. The system can be used to detect S. aureus in farm wastewater to provide the accurate data for choosing the method of wastewater treatment. The system plays a positive role in water, solid aquaculture pollution control by real-time detection the S. aureus in farm wastewater.The two system established in this thesis can be used to rapid, accurate, real-time detect S. aureus with high specificity, even though the principles of the two system are different. These two systems can be used to confirm each other by detecting the same sample. The microfluidic-LAMP assay is more sensitive which is more suitable for low S. aureus concentration detection.This work is a cooperation project of the department of environmental science and engineering and institutes of biomedical sciences. Realize rapid, accurate, real time detect S. aureus which may cause contamination through the combination of, molecular biology, immunology and microfluidic technologies. It plays a positive role in S. aureus pollution control and is of great significance in contamination control, environment safety, human health, biosafety guarantee, environmental warning. |
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