【作者】 李滨；

【导师】 陈伟；

【作者基本信息】 合肥工业大学 ， 水产品加工及贮藏工程， 2022， 硕士

【摘要】 近年来,随着人们对生活品质要求的提高,“轻食主义”开始盛行,特别是像蔬菜沙拉、水果沙拉和刺身拼盘(如鱼、虾等)等,渐渐获得人们的喜爱。致病菌污染是蔬果以及水产品中最常见的问题,像沙门氏菌和金黄色葡萄球菌等食源性致病菌的污染。而直接食用被致病菌感染的食物,会造成严重的身体伤害,所以需要相应的手段防患于未然。提前检出对于预防致病菌感染极其重要,在这里我们提出了几种简单快速的检测方法,可用于现场快速检测。(1)基于酶促重组等温扩增(ERA)结合胶体金试纸条检测沙门氏菌本章主要以蔬菜及水产品作为检测样本,研究了蔬菜表面致病菌的富集方法,DNA提取的手段,以及试纸条检测的优化。采用过滤的方式富集蔬菜表面的致病菌,再通过5 min的超声便可以获得目标物的DNA,经过20 min的等温扩增获得扩增产物,最后试纸条3 min展示结果,总检测时长仅为40 min,检测范围为10~7-10~2CFU/m L,这对于现场快速检测非常重要。(2)基于链交换等温扩增(SEA)结合荧光试纸条检测金黄色葡萄球菌本章主要优化了链交换等温扩增的反应体系以及反应条件,整个扩增反应过程可以在40 min完成,优化了荧光试纸条的显色条件,极大地提高了检测灵敏度,检测范围为10~7-10~1CFU/m L。实验表明,该方法对金黄色葡萄球菌的检测是非常灵敏的。(3)基于SEA等温扩增结合乳胶试纸条双重检测沙门氏菌和金黄色葡萄球菌本章主要从多重检测的方向考虑,在引物端修饰了小段DNA单链作为杂交探针,保证双重检测的特异性。优化了双重扩增引物的添加量,降低两者引物之间的竞争关系;优化了试纸条的显色条件,提高了检测的灵敏度以及特异性。对两种致病菌的检测浓度可达到10~2和10~3CFU/m L,这表明本实验对于复杂样品的检测具有优势。更多还原

【Abstract】 In recent years,with the improvement of people’s requirements for the quality of life,"Light Food,Healthy Diet"has become a popular eating habit.As a result,vegetables platters,fruit salads and sashimi plates(such as fish and prawn etc.),which are loved by people.The most common problem in vegetables,fruits and aquatic products is pathogen contamination,such as Salmonella,Staphylococcus aureus and so on.Ingesting food infected with pathogenic bacteria will pose a great threat to human health,so corresponding measures are needed to prevent microbial contamination of food.Early rapid screening is extremely important for preventing pathogenic bacteria.Here,we propose several simple and rapid detection methods that can be used for rapid on-site detection of pathogenic bacteria.(1)Detection of Salmonella based on enzymatic recombinant isothermal amplification(ERA)combined with colloidal gold test stripsIn this chapter,vegetables and aquatic product are selected as the test samples.Through enrichment,nucleic acid extraction and amplification of pathogenic bacteria on the surface of vegetables,and optimization of the detection conditions of lateral chromatography test paper,a rapid and sensitive detection method for Salmonella in food is finally established.The pathogenic bacteria on the surface of vegetables are enriched by filtration,and then the DNA of the target can be obtained by 5 min of ultrasound,and the amplified product was obtained after 20 min of isothermal amplification.Finally,the lateral flow strip displayed the results for 3 min.The whole process takes only 40 min to complete,and the detection linear range is 10~7-10~2CFU/m L.Our research provides new ideas for rapid detection of food safety.(2)Detection of Staphylococcus aureus based on strand exchange isothermal amplification(SEA)combined with fluorescent test stripsIn this chapter,we mainly optimized the reaction parameters of the system in the strand-exchange isothermal amplification.The entire amplification reaction process can be completed in 40 min.After the prioritization of the fluorescent test strips,the detection sensitivity is greatly improved.The detection linear range of this method was10~7-10~1CFU/m L.All of the test results show that the proposed method has high sensitivity for Staphylococcus aureus.(3)Double detection of Salmonella and Staphylococcus aureus based on enzymatic recombinant isothermal amplification combined with latex test stripsIn this chapter,we mainly focus on the multiplex detection.A small DNA strand is modified at the primer end as a hybridization probe to ensure the specificity of dual detection.The added amount of double amplification primers is optimized to reduce the competition relationship between the two primers;the experimental parameters of the test strip are also optimized,which improves the detection sensitivity and specificity.The detection concentration of pathogens can reach 10~2or 10~3CFU/m L,which is especially important for the detection of complex samples.更多还原