【作者】 张超；

【导师】 栗绍文；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 硕士

【摘要】 单核细胞增生李斯特菌(Listeria monocytogenes,LM)是李斯特菌属中唯一对人致病的胞内寄生菌,能引起老人、孕妇等免疫力低下人群严重的胃肠道、全身系统性和中枢神经系统的食源性感染,是一种世界公共卫生领域重要的食源性病原菌。2005年-2013年国内外LM污染事件不断频发,因此需要建立快速准确的LM检测方法。本研究选取LM的正向转录调控因子A (prfA)基因为靶基因,建立了LM的PCR和HNB-LAMP检测方法。1.LM的prfA基因的克隆分析根据GenBank中收录的LM的prfA基因序列,应用分子生物学软件Primer Premier 5.0设计特异性引物来扩增目的基因。测序分析显示LM模式菌株和临床分离菌株的prfA基因与LML312等菌株的prfA基因均具有99%的核苷酸同源性。2.PCR法和HNB-LAMP法的建立设计引物并通过优化反应体系和反应条件建立了PCR和LAMP检测方法。在LAMP反应体系中加入显色剂羟基萘酚蓝(HNB)建立了阳性显天蓝色、阴性显紫罗兰色,可目视结果的HNB-LAMP法。特异性和灵敏度试验结果表明本研究建立的PCR方法和HNB-LAMP方法均能快速准确检测出LM模式菌株和临床分离株,而对EHEC O157:H7、沙门氏菌、金黄色葡萄球菌等食源性病原菌的检测结果均为阴性。3.PCR法和HNB-LAMP法的初步应用1mL浓度为4.5CFU/mL菌液人工污染猪肉样品,经增菌培养16h后PCR法可检出LM,经增菌培养8h后LAMP法可检出LM。用国标法检测采集的200份临床冷鲜猪肉样品,分离的51株LM疑似菌株只有3株为LM阳性,48株阴性为非LM李斯特菌,LM的阳性率达1.5%。同时PCR法和HNB-LAMP法检出的LM阳性菌株与国标法分离检出的LM阳性菌株一致,检测结果符合率达100%。更多还原

【Abstract】 Listeria monocytogenes is one of human intracellular pathogenic bacteria in Listeria.spp, and can cause the serious gastrointestinal, systemic and neutral infection of people with low immunity. It is an important foodborne pathogen to public health in the world. LM pollution events frequently emerged from 2005 to 2013, so fast and accurate LM detection methods must be developed. In this study, PCR and HNB-LAMP methods for detection of LM were developed by using LM positive transcriptional regulatory factor A (prfA) gene as target gene.1.Cloning and analysis of prfA gene of LMLM positive transcriptional regulatory factor A (prfA) genes were amplified by using the special primers based on the referencing gene sequence in GenBank and designed by using the molecular biology software Premier 5.0.Sequencing analysis showed that the prfA genes of LM model strain CCTCCAB97021 and clinical isolate strain had 99%of nucleotide sequence similarity with that of the referencs serotype 4b strains LM L312.2 Development of PCR and HNB-LAMP methodsIn this study PCR and LAMP methods for detection of LM were developed by optimizing the reaction system. Hydroxy naphthol blue (HNB) was added to the LAMP reaction system as a color-developing agent and so HNB-LAMP method was visible. The specificity and sensitivity results showed that PCR and HNB-LAMP methods could be used to quickly and accurately detect LM model strain and clinical isolate. While the detection results of EHEC 0157:H7, Salmonella, Staphylococcus aureus and other foodborne pathogens were negative.3. The application of PCR and HNB-LAMP methodsThe pork was artificially polluted with the 4.5 CFU/mL concentration of LM culture. PCR method could detect LM after the samples were cultured for 16h., but LAMP method could detect LM after the samples were cultured for 8h. This study detected 100 clinical cold fresh pork samples by using standard isolation and identification method.51 suspected strains were isolated, in which 3 strains were LM and 48 strains were non-LM Listeria spp. At the same time the isolated strains were also detected by PCR and HNB-LAMP methods. The results were consistent with the standard isolation and identification method, which showed the PCR and HNB-LAMP methods had higher accuracy rate.更多还原