臺灣博碩士論文加值系統

In Taiwan, among various staphylococcus enterotoxins (SEs) produced by Staphylococcus aureus, SEA is the most related to food poisoning outbreaks, followed by SEB. In the United States, annually there are 185,000 cases of foodborne illnesses caused by SEs. Until now, more than 20 kinds of SEs have been reported. SEs have the superantigenic activities and can resist to heat and proteases in human gastrointestinal tract. The syndromes caused by SEs include diarrhea, vomiting, retching, abdominal cramping and prostration, and a toxin dose of less than 1 μg will cause these symptoms. Therefore, the purpose of this study is to develop rapid assays for detecting enterotoxic S. aureus in food. First, we developed an immunomagnetic bead (IMB) based method with the use of NeutrAvidin-tagged liposomal nanovesicles (NA-LNs) which encapsulated fluorescent dyes as the detection reagent to detect S. aureus containing sea gene. Through a PCR reaction, the target DNA was amplified and labeled with digoxigenin (Dig) and biotin. The amplified target DNA was then captured by IMB modified with anti-Dig-antibody and detected by NA-LNs. The developed assay could detect S. aureus and differentiate it from Salmonella enterica and Escherichia coli, with a limit of detection (LOD) of 101 CFU mL-1 without pre-enrichment. With a 2-hour pre-enrichment, this developed assay could detect as little as 1 CFU in 25 mL of milk. Furthermore, we developed a multiplex loop-mediated isothermal amplification (m-LAMP) combined with a lateral flow assay (LFA) for simultaneously detecting the sea and seb genes of enterotoxic S. aureus. The LOD of this assay was 102 CFU mL-1 S. aureus, which was 10-fold lower than that of a multiplex PCR; and this assay did not show any cross-reactivity as detecting other enterotoxic S. aureus strains or other food pathogens. After 4~6-hour enrichment, this developed assay could detect as low as 1 CFU mL-1 of S. aureus in four different food matrixes - milk, apple juice, cheese, and rice. Conclusively, these two developed methods can be completely finished within a workday, which can provide an alternative way to easily and quickly screen the contamination of enterotoxic S. aureus in food products or in food supply chains.

Chinese Abstract i
English Abstract ii
List of Figures v
List of Tables vii
Abbreviations Table viii
1. Literature Review 1
1.1. Staphylococcus aureus 2
1.2. Staphylococcal food poisoning 4
1.3. Staphylococcal enterotoxin 7
1.4. Detection of Staphylococcus aureus or staphylococcal enterotoxin 13
1.5. Loop-mediated isothermal amplification 18
1.6. Lateral flow assay 20
1.7. Magnetic bead 24
1.8. Liposomal nanovesicles 26
2. Dual-labeled PCR-based immunofluorescent assay for the rapid and sensitive detection of enterotoxic Staphylococcus aureus using cocktail sized liposomal nanovesicles as signal enhancer 28
2.1. Abstract 29
2.2. Introduction 30
2.3. Materials and Methods 33
2.3.1. Materials 33
2.3.2. Preparation of immunomagnetic beads 33
2.3.3. Preparation of Neutravidin-tagged liposomal nanovesicles 34
2.3.4. PCR reaction 34
2.3.5. Bacterial growth 35
2.3.6. Preparation of contaminated food 35
2.3.7. Assay optimization and performance 35
2.3.8. Statistical analysis 37
2.4. Results and Discussion 38
2.4.1. Optimization of assay format 38
2.4.2. Effect of biotinylation position on assay signal 39
2.4.3. Optimization of IMB reaction conditions 40
2.4.4. Optimization of NA-LNs 41
2.4.5. Performance of IMB/ LNs fluorescence 43
2.5. Conclusion 45
3. Combined multiplex loop-mediated isothermal amplification with lateral flow assay (m-LAMP/LFA) to detect sea and seb genes of enterotoxic Staphylococcus aureus 55
3.1. Abstract 56
3.2. Introduction 57
3.3. Materials and Methods 59
3.3.1. Materials 59
3.3.2. Multiplex LAMP assay 59
3.3.3. Conventional multiplex PCR 59
3.3.4. Preparation of LFA strips and a format of m-LFA 60
3.3.5. Preparation of food sample 60
3.4. Results and Discussion 62
3.4.1. Optimization of m-LAMP 62
3.4.2. Optimization of multiplex LFA 62
3.4.3. Specificity and validation of the m-LAMP/LFA 64
3.4.4. Sensitivity of m-LAMP/LFA 64
3.4.5. Analysis of food sample 65
3.5. Conclusions 65
4. Conclusions 74
References 77

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