【作者】 蔡潭溪；

【导师】 黄克和；

【作者基本信息】 南京农业大学 ， 临床兽医， 2006， 硕士

【摘要】 副溶血弧茵(Vibrio Parahaemolyticus)和创伤弧菌(Vibrio vulnificus)是引发食源性疾病的重要病原菌。研究数据表明，50％～70％因食用海产品引发的腹泻病例是由副溶血弧菌引起的。创伤弧菌通过进食生的牡蛎穿过胃肠道黏膜或通过破损的皮肤接触海水入血而感染，并在短时间内出现败血症和蜂窝组织炎、出血性大疱，一旦出现败血症，其死亡率高达60％。因此，副溶血弧菌和创伤弧茵对人的健康是一种潜在危险，检查食品中副溶血弧菌和创伤弧菌及数量具有重要的实际意义。 荧光定量PCR技术根据荧光能量传递原理，融合了PCR技术的高效扩增、探针技术的高特异性、光谱技术的高敏感性和高精确性等优点，可直接探测PCR过程中荧光信号的变化获得定量结果。根据荧光定量PCR多通道激发光源，多个滤光片的检测体系以及标记不同荧光染料的探针，可达到同时检测两种或更多种细菌的目的，为同步定量检测食品中的副溶血弧菌和创伤弧菌提供了必要的条件。 本文建立了用单一Real-time PCR技术定量检测副溶血弧茵和用双重Real-time PCR技术同步定量检测副溶血弧菌和创伤弧菌的新方法，并用该方法及传统的培养方法对华东地区海产品中副溶血孤菌和创伤弧菌的污染情况进行了调查。 试验Ⅰ从单一Real-time PCR技术入手，根据GenBank公布的副溶血弧菌的gyrB基因序列设计一对引物和TaqMan探针，建立了基于TaqMan探针的Real-timePCR方法。通过对9种细菌(12株菌株)的DNA进行扩增，结果所有4株副溶血弧茵均可产生扩增曲线，其他8株非副溶血弧茵均不产生扩增曲线，证明了引物和探针具有很高的特异性。细菌纯培养液和人工布菌样品的检测敏感度分别为1 CFU/PCR反应体系和10 CFU/PCR反应体系，相关系数均为0.99(r~2=0.99)。整个试验可于1h内完成。该试验建立的方法可快速定量检测海产品种的副溶血弧菌。 试验Ⅱ针对副溶血弧菌的gyrB基因序列和创伤弧菌的vvh序列分别设计引物和TaqMan探针，建立双重Real-time PCR检测体系，制作标准曲线，同步定量检测副溶更多还原

【Abstract】 Virio parahaemolyticus and Vibrio vulnificus are two of pathogenic vibrio species primarily involved in causing gastrointestinal illnesses. V. parahaemolyticus is considered to be the causative agent in 50% to 70% of all cases of diarrhea associated with the consumption of fishery products in the summer months. Vibrio vulnificus produces a rapidly fatal septicemia, with a mortality rate of up to 60%, which is primarily associated with the ingestion of raw oysters. Contact of wounds with seawater or shellfish can also lead to serious infections that can progress to septicemia. Therefore, the detection of the presence and the number of V.parahaemolyticus and V. vulnificus were essential.The multiplex Real-time PCR system uses fluorogenic probes to detect PCR products as they form; the exonuclease activity of Taq polymerase releases a labeled reporter dye at the 5’ end of the probe from the quencher dye at the 3’ end with each cycle of amplification. Thus, increased fluorescence is directly proportional to the formation of PCR products. Plotting the increase in fluorescence versus cycle number gives a comprehensive picture of the PCR process, and quantification of initial template concentration can be calculated from data on the exponential phase of amplification. Multiplex Real-time PCR offers rapid, sensitive, and specific detection V. parahaemolyticus and V. vulnificus.In present paper, a new technique based on real-time PCR for quantitative detection V. parahaemolyticus and a multiplex real-time PCR for simultaneous and quantitative detection of Vibrio parahaemolyticus and Vibrio vulnificus were established. Then a screening of seafood in Eastern China for the presence of Vibrio parahaemolyticus and Vibiro vulnificus was carried out base on the mutiplex real-time PCR and Culture Method. 1 Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus Vibrio parahaemolyticus is one of important human food pathogens. Traditional diagnostic tests for V. parahaemolyticus are laborious and aways present flase negative更多还原