节点文献
影响食品安全的食源性致病菌快速检测技术与风险分析研究
Studies on the Detection Protocols and Risk Anlysis to Food Borned Pathogen for the Food Safty
【作者】 邱驰；
【导师】 段玉玺；
【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2008， 博士
【摘要】 食品安全问题一直是各国政府和人民群众最为关心的问题,它关系到消费者的健康和生命。由病原细菌引起的食源性疾病是影响食品安全的最主要的因素之一。因此对食源性致病菌快速、准确的检测问题亟待解决。本研究针对在食品微生物污染中比较常见的致病菌,开展了以分子免疫学、分子生物学为基础的检测技术改进与应用研究,采用胶体金免疫层析法,常规PCR法、实时荧光PCR法、高通量基因芯片法对食源性致病菌进行检测的系统研究,并把这些检测技术首次应用于进出境商品的鉴定检测,获得了成功。首次系统地建立了国内外第一个食源性致病菌的检测技术体系,该体系的成功建立,极大的提高了进出境食品的检验效率,缩短了检验时限,对突破国外对我国设置的技术贸易壁垒起到了关键作用。利用胶体金免疫层析技术,对金黄色葡萄球菌A、B型肠毒素,单增李氏菌溶血素O进行了快速检测的研究,开发了具有我国自主知识产权的葡萄球菌肠毒素和单增李斯特菌及李斯特菌溶血素胶体金检测试剂条,建立了一套方便现场使用并且容易操作的检测方法。该方法特异性强,不需任何附加设备,检测时间大为缩短,灵敏度高。为食源性致病菌的前期快速筛选做好了技术储备。通过常规PCR技术对食品中常见的病原细菌进行了快速检测的试验结果表明,应用PCR方法可以很好的对食品中的食源性病原菌进行检测,仅用几个小时即可完成检验过程,大大的缩短了检测时间。对于缩短进出境货物通关时间具有十分重要的意义。本研究采用实时荧光PCR技术,针对沙门氏菌等食品中常见的致病菌,通过检索文献,确定致病菌的靶基因序列,利用生物软件进行序列对比,截取最一致的序列进行引物探针设计,并筛选出适合实时荧光PCR检测的特异性探针,建立了沙门氏菌等11种食源性致病菌的实时荧光PCR技术,并研制出了适合于进出口检验检疫、疾病控制、临床诊断及产品质量检验使用的试剂盒。通过实时荧光PCR法检测食源性致病菌与国家标准、行业标准方法的比较,检出率完全相同,显示出该方法具有广泛而良好的适用性,对于11种食源性致病菌的检测限度可以达到84 cfu/mL-7000 cfu/mL之间完全满足检验检疫业务的需要。并且首次在DNA模板的制作过程中做出了平板菌落煮沸法的尝试,节省了试验步骤,取得了较好试验效果。在基因芯片高通量检测食源性致病菌的研究中,优化出反应体系的9条探针及引物位点,并使多重PCR能够做到多重引物的同时扩增,克服了多重PCR反应条件优化过程中各引物之间的相互干扰,以及引物、探针荧光标记产物的长度和结构之间等存在的相互干扰等技术难点,确定了55℃为最适合杂交温度以及最佳杂交液配置方案,从而建立了基于多重PCR技术的高通量检测食源性致病菌的基因芯片检测技术。对丹东口岸入境的十种冷冻水产品引起李斯特菌病、四种冷冻水产品引起沙门菌病和六种冷冻水产品引起副溶血性弧菌胃肠炎疾病的风险进行评估。建立了定量食源性致病菌危险性评估模型,结果表明:单次消费经丹东口岸进口的冷冻章鱼、海螺、青柳蛤肉、香螺、江瑶贝、蟹子、河虾、河螺肉、紫石房蛤和其它贝类,引起李斯特菌病的概率几乎为零,不具有危险性。单次消费经丹东口岸进口的冷冻海螺、河螺肉、香螺和其它贝类,引起沙门菌病的概率,对于正常人群分别为1.57×10-2、7.62×10-3、2.19×10-2和1.62×10-2;对于易感人群分别为0.12、6.61×10-2、0.15和0.12。单次消费经丹东口岸进口的冷冻海螺、章鱼、河螺肉、紫石房蛤、江瑶贝和蟹子,引起副溶血性弧菌胃肠炎的概率,分别为6.21×10-6、6.65×10-7、3.85×10-7、1.26×10-8、1.28×10-6和9.18×10-7。本研究首次建立了食源性致病菌分子免疫学、常规PCR、实时荧光PCR和基因芯片检测以及同国家标准比对的技术体系,这也是国际上首次研究建立的的食源性致病菌的检测技术体系。本研究还在国际上首次对朝鲜进境的冷冻水产品进行了风险评估。
【Abstract】 Food safety was always concerned by governments and the people.It related the health and life of consumers.Food borne pathogens were the primary reasons leading to tbod poisoning.Thus,the detection for food borne pathogens was urgently needed.This research is based on common pathogenic pathogens in food,the molecular immunology,the molecular biology detection approaches were carried out.In the research,the immunity chromatographic analysis,PCR,the Real-time fluorescence PCR,the genetic chip were used for food borne pathogens detection.And these detection techniques were firstly applied to imported and exported commodity inspection.The newly developed detection protocols were for the first time in the world utilized to detect food borne pathogens.Which have momentous meanings for improving the imported and exported food inspection efficiency,reduce the time of inspection and remove the trade barriers which constituted by developed nations.The immunity chromatographic analysis technology was used for detecting staphylococcal enterotoxin A,B and C and Listeriolysin O.By using this technology, the inspection boxes having our own intellectual property rights were developed,and a set of rapid field inspection method was established.The method is specificity and more efficient than conventional methods.The establishment of this detection method provided a firm technical and theoretical basis.To detect food borne pathogens rapidly,the PCR technology was performed in this study.The results show that the PCR technology was availability for detecting food borne pathogens.The PCR method can greatly reduce the detection time,which have momentous meanings for cargo going through customs immediately.General food borne pathogens,such as the Salmonella,were known using the real-time fluorescence PCR approach to detect.By searching literature and using the isogenous sequence similarity BLAST analysis,the food borne pathogens target gene sequences were found.Based on these sequences,specific primers and probes for real time PCR detection were designed and selected.The systematically compared results between real time PCR method and GB,SN were identical.From these studies,the real time fluorescence PCR method is established.And the detection boxes were applied to imports and exports inspection,disease control and clinical diagnosis.The limit of detection was approximately 84cfu/mL -7000cfu/mL.In DNA extract experiment,the results of using boiled Salmonella enrichment broth and boiled single Salmonella bacterium as template of the real time PCR were as good as using Salmonella DNA. In the study of genetic chip of detection food borne pathogens,9 primers and 9 probes which are uniform in PCR detection profile and condition were selected and optimized,which could primers in one multiple PCR reaction to perform amplifying reaction simultaneously,so the interaction interferences between primer pairs in the process of multiple PCR reaction condition optimization,between every pairs of primers and products of multiple PCR reaction and between oligonucleotide length and structure of primers,probes and fluorescein labeled products were overcome.The studies showed 55℃was the best hybrid temperature for gene hybrid.Thus the protocols of high density food borne pathogens detection chip based on multiple PCR technique were established.The risk analysis of frozen aquatic products from Dandong port was carried out. The risk analysis mathematic model of food borne pathogens was established.The results showed that the risk probability of Listeria monocytogenes that take the frozen octopus,conch and crab,etc.for once were zero.The risk probability of Salmonella was 1.57×10-2,7.62×10-3,2.19×10-2 and 1.62×10-2 to health people.The Salmonella risk probability was 0.12,6.61×10-2,0.15 and 0.12 to susceptibility people. The risk probability of V.parahaemolyticus was 6.21×10-6,6.65×10-7,3.85×10-7,1.26×10-8,1.28×10-6 and 9.18×10-7.In this research,the food borne pathogens detection protocols based on molecular immunology,PCR,the real time PCR and gene chip were established for the first time in the world.And the risk analysis of frozen aquatic products from the North Korea was firstly carry out in the world.