【作者】 邱晋；

【导师】 裴晓方；

【作者基本信息】 四川大学 ， 营养与食品卫生学， 2007， 硕士

【副题名】自制裸磁珠吸菌性能研究和实时荧光PCR检测副溶血性弧菌方法的建立

【摘要】 目的：研究自制裸磁珠对常见食源性致病菌的吸附性能；建立TaqMan—实时荧光PCR快速检测副溶血性弧菌的方法，用于食物中毒快速诊断和食品中副溶血性弧菌的污染状况调查。方法：第一部分自制裸磁珠吸菌性能研究1．磁珠对低菌量菌液的吸附与浓集的研究研究磁珠对低菌量细菌的吸附性能、温度对磁珠吸附性能的影响及磁珠对样品中细菌的吸附性能。2．磁珠对高菌量菌液的吸附与浓集的研究研究高菌量时磁珠对实验菌的吸附方法、干式和湿式磁珠对细菌的吸附性能、不同磁珠用量对细菌的吸附、原始菌量对磁珠吸附容量的影响、不同孵育时间对实验菌的吸附以及不同孵育温度对实验菌的吸附情况。第二部分TaqMan—实时荧光PCR快速检测副溶血性弧菌方法的建立1．引物和探针设计根据GenBank数据库中副溶血性弧菌tdh基因的保守序列设计引物和探针，并提交BLASTN进行比对，探讨其代表性。2．TaqMan—实时荧光PCR检测目的菌方法的建立与反应条件优化建立基于TaqMan—实时荧光PCR快速检测副溶血性弧菌反应体系并进行反应条件优化以及灵敏度、特异性试验。3．未知菌量菌液浓度测定以模板菌浓度的对数值为横坐标，Ct值为纵坐标绘图，制作标准曲线，定量测定未知菌量的副溶血性弧菌。结果：1．自制裸磁珠对低菌量菌液吸附：37℃对常见食源性致病菌和卫生指示菌有很强的吸附，吸附率达到97％。食品成分对其吸附有一定影响，吸附率最低为58．42％。2．自制裸磁珠对高菌量菌液吸附：磁珠用量与吸附率近似正相关，吸附容量与原始菌量存在对数线性相关。温度影响吸附效率。吸附作用40min后吸附率无明显差异，达到平衡。此外，干式和湿式保存的磁珠，对试验细菌的吸附率无明显差异。3．TaqMan—实时荧光PCR快速检测体系灵敏度高，能达到1．3×10~4cfu／ml(33cfu／PCR反应体系)，对8株携带tdh基因的副溶血性弧菌能检测到荧光信号，而对其它弧菌菌株则检测不到荧光信号，无交叉反应，特异性好，可用于食物中毒快速诊断和食品中副溶血性弧菌的污染状况调查。结论：磁分离技术不仅能快速吸附和浓集细菌，而且能同时吸附多种目的菌，通过实验自制裸磁珠对不同菌量的菌液都有比较理想的吸附。实时荧光PCR技术的快速定量检测是其它检测方法无法比拟的，其较低的检出限、良好的特异性和实验时间短是这一技术的主要特点，用于副溶血性弧菌的快速检测灵敏度较高，特异性强，可用于副溶血性弧菌的快速定量检测。所以把磁分离技术和实时荧光PCR技术应用于副溶血性弧菌食物中毒的快速诊断和食品污染状况调查方面具有广阔的前景。更多还原

【Abstract】 Objective:To study the adsorption capacity of lab made magnetic beads to the common food-bome pathogenic bacteria; to establish a method of rapid detection of Vibrio parahemolyticus (VP) using TaqMan real-time PCR for the Vibrio parahemolyticus food poisoning diagnosis and the investigation of Vibrio parahemolyticus contamination.Methods:Part One Study on the adsorption capacity of lab made magnetic beads1. Study on the adsorption capacity of lab made magnetic beads to the low number of bacteria in samplesStudy on the adsorption capacity of lab made magnetic beads to the different bacteria, at different temperature and tothe bacteria in different samples.2. Study on the adsorption capacity of lab made magnetic beads to the high number of bacteria in samplesStudy on the different adsorption methods of lab made magnetic beads to the bacteria, on the adsorption capacity of dry and wet lab made magnetic beads to the bacteria, the adsorption capacity of different amount of lab made magnetic beads to the bacteria, the adsorption capacity to the different high number of bacteria, in different reaction time, and at the different temperature.Part Tow Establish a method of rapid detection of Vibrio parahemolyticus using TaqMan real-time PCR1. Primers and probe designDesign the primers and TaqMan probe based on the sequence of tdh gene published on GenBank, estimate their feasibilities with BLASTN.2. Optimization of the PCR conditionsEstablish a rapid quantitative detection method for Vibrio parahemolyticus using TaqMan real-time PCR, optimize the PCR conditions, and analyze its sensitivity and specificity.3. Detection of the target bacteriaPlot the log cell number versus the TaqMan real-time PCR cycle threshold for 10 fold dilutions of template, fix quantify of the target bacteria.Results:1. The adsorption capacity of lab made magnetic beads to the low number of bacteria in samples: The lab made magnetic beads had great adsorption capacity to bacteria at 37℃; the adsorption efficiency could reach up to 97％. The component of the tested samples could decrease the adsorption capacity of the magnetic beads, and the adsorption efficiency decreased to 58.42％.2. The adsorption capacity of lab made magnetic beads to the high number of bacteria in samples: The adsorption capacity was correlative with the amount of the magnetic beads. The adsorption capacity was correlative with the number of the bacteria in orders. The temperature was an influential factor of the adsorption capacity of the beads. The adsorption capacity increased as the incubate time extend until to 40 minutes. The preservation method for beads whould likely not an obvious factor to influence the adsorption.3. The TaqMan real-time PCR assay is rapid, sensitive (1.3×10~4cfu/ml、33cfu/PCR reaction) and specific. It could be applied to the rapid diagnosis of Vibrio parahemolyticus food poisoning and investigation of the contamination status of it in food.Conclusions:The magnetic separation technology can rapid adsorb and enrichment the bacteria. The adsorption capacity of lab made magnetic beads to the bacteria in different numbers is quite well. Comparison of other detection methods, the TaqMan real-time PCR assay is rapid, sensitive and specific. The combination of magnetic separation technology and the TaqMan real-time PCR assay could be applied to the rapid diagnosis of Vibrio parahemolyticus food poisoning and investigation of the contamination status of it in food更多还原