2013年-2016年淄博市副溶血性弧菌血清分型、分子特征及溯源研究

发布时间：2018-05-23 06:39

本文选题：副溶血性弧菌 + 生化分型　；参考：《青岛大学》2017年硕士论文

【摘要】：副溶血性弧菌(V.parahaemolyticus,VP)是嗜盐性革兰氏阴性菌,主要存在于我国沿海区域,该菌能够引起食源性食物中毒及疾病,从而导致地方性流行的胃肠炎暴发,其发生规模在我国沿海和部分内陆区域已经表现出明显的上升趋势,高居细菌性食物中毒的首要位置。大量研究发现,耐热性直接溶血素(thermostable direct hemolysin,TDH)或TDH相关溶血素(TDH-related hemolysin,TRH)存在于能够致病的副溶血弧菌中。这两种溶血素是导致副溶血性弧菌致病的主要毒力因子,与该菌的致病能力密切相关。副溶血性弧菌实验室的常用分型方法主要有生化分型和血清学分型,这两种方法不能有效地对目标菌的同源性进行分析,但是脉冲场凝胶电泳技术(Pulsed field gel electrophoresis,PFGE)方法具有分型力好、分辨力强、可重复性的优点,常被用于微生物的鉴定分型研究。本研究对淄博市2013年-2016年自水产品分离出的副溶血性弧菌菌株毒力相关基因的分布情况进行了分析,同时应用PFGE技术对VP分离菌株进行分子分型,研究其同源性,为副溶血性弧菌导致食源性疾病的防控提供实验依据。目的:利用血清分型方法、毒力基因分子检测和PFGE技术对淄博市2013年-2016年市售水产品中分离得到的副溶血性弧菌进行比对分析,了解近4年该目标菌之间存在的关系,分析其流行规律,为有效预防和控制食源性疾病的发生提供科学依据。方法:对淄博市2013年-2016年分离自市售新鲜水产品的副溶血性弧菌菌株进行复苏培养和复核鉴定。对上述目标分离菌株进行血清分型,探讨血清型的分布特点;用PCR对tdh和trh基因进行检测;PFGE技术进行副溶血性弧菌的分子分型,探讨菌株的同源性。结果:(1)85株分离株经复核鉴定,复核结果与初始鉴定一致。共分出7个O抗原血清型,以O2群为优势血清型,占29.41%。K抗原鉴定出型别较少,只有5种,且分布较分散,无明显趋势。(2)多重荧光PCR检测结果表明,85株分离菌株毒力相关基因tdh携带率为3.53%,trh携带率为5.88%,trh高于tdh。(3)PFGE分型结果显示,血清型同为O2:K28的2株分离株PFGE图谱不尽相同,其他分离菌株血清型各不相同且PFGE图谱各自成型,相似度极低。携带毒力基因的分离株间亲缘关系较远,没有明显优势带型。结论:(1)淄博市副溶血性弧菌分离菌株O抗原分布以O2群居多,K抗原型别较少且分布较分散,无明显趋势。(2)市售水产品分离菌株中,毒力基因tdh和trh检出率较低,且均只携带单一毒力基因。(3)本次实验水产品中副溶血性弧菌的血清型和PFGE图谱类别相关性不明显,PFGE带型分散,其亲缘关系较远。
[Abstract]:Vibrio parahaemolyticus (V. parahaemolyticus) is a halophilic gram-negative bacteria, which mainly exists in coastal areas of China. It can cause foodborne food poisoning and diseases, thus leading to the outbreak of endemic gastroenteritis. Its occurrence scale has shown an obvious upward trend in coastal and some inland areas of China and occupies the primary position of bacterial food poisoning. A large number of studies have found that thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH) are present in vibrio parahaemolyticus which can cause the disease. These two hemolysins are the main virulence factors leading to virulence of Vibrio parahaemolyticus and are closely related to the pathogenicity of Vibrio parahaemolyticus. The commonly used typing methods of Vibrio parahaemolyticus laboratory mainly include biochemical typing and serological typing. These two methods can not effectively analyze the homology of the target bacteria, but pulsed field gel electrophoresisPFGE has good typing power. The advantages of strong resolution and repeatability are often used in the identification and typing of microorganisms. In this study, the distribution of virulence related genes of Vibrio parahaemolyticus isolated from aquatic products in Zibo City from 2013 to 2016 was analyzed. At the same time, the virulence genes of Vibrio parahaemolyticus strains isolated from aquatic products from 2013 to 2016 were genotyped by PFGE to study their homology. To provide experimental basis for the prevention and control of foodborne diseases caused by Vibrio parahaemolyticus. Objective: to compare and analyze Vibrio parahaemolyticus isolated from aquatic products sold in Zibo city from 2013 to 2016 by using serotyping method, virulence gene molecular detection and PFGE technique, and to understand the relationship between Vibrio parahaemolyticus and Vibrio parahaemolyticus in recent 4 years. The analysis of its epidemic law provides scientific basis for effective prevention and control of foodborne diseases. Methods: Vibrio parahaemolyticus strains isolated from fresh aquatic products from 2013 to 2016 in Zibo City were resuscitated and identified. The serotyping of the target isolates was carried out, and the distribution of the serotypes was discussed. The molecular typing of Vibrio parahaemolyticus was carried out by using PCR to detect the tdh and trh genes, and the homology of the strains was discussed. Results the results were consistent with the initial identification. A total of 7 O antigen serotypes were identified, with O2 group as the dominant serotype, accounting for 29.41.K antigens, only 5 types of O antigen serotypes were identified, and their distribution was scattered. The results of multiplex PCR analysis showed that the carrying rate of virulence related gene tdh was 3.53 and 5.88trh was higher than that of tdh.(3)PFGE typing. The results showed that the PFGE patterns of the two isolates with the same serotype of O2:K28 were different. The serotypes of other isolated strains were different and the PFGE profiles were formed respectively, and the similarity was very low. The isolates carrying virulence genes had a long genetic relationship, and there was no obvious dominant band type. Conclusion the distribution of O antigen of Vibrio parahaemolyticus isolates in Zibo city is less and more scattered than that of O 2 colony, and there is no obvious trend. The detection rate of virulence gene tdh and trh is lower in the isolated strains of aquatic products on the market. The serotype of Vibrio parahaemolyticus in aquatic products was not significantly correlated with the type of PFGE profile.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155.5

【参考文献】