单增李斯特菌两种快速检测方法的建立及其试剂盒的研制

发布时间：2018-03-30 01:39

  本文选题：单核细胞增生性李斯特菌　切入点：环介导等温扩增　出处：《吉林大学》2017年硕士论文

【摘要】：单核细胞增生性李斯特菌是一种食源性人兽共患病的革兰氏阳性病原菌,可引起人和多种动物罹患李斯特菌病。早在20世纪90年代,它已被列为四种食源性致病菌之一,近年来,单核细胞增生性李斯特菌感染病例在世界范围内呈上升趋势。目前,该细菌的传统检测方法,操作流程复杂,检测周期长,难以满足快速检测食品所需效率。因此,有必要建立一种快速、方便的单核细胞增生性李斯特菌的检测方法。本研究针对单核细胞增生性李斯特菌(以下简称单增李斯特菌)hly A基因设计了4条特异性的环介导等温核酸扩增(loop-mediated isothermal amplification,LAMP)引物,通过筛选引物、优化反应条件,建立了单增李斯特菌的LAMP快速检测方法。试验结果显示,该方法能够特异地检测单增李斯特菌,其最低检测限为3 CFU/m L。扩增产物可通过琼脂糖凝胶电泳、显色反应进行判定。LAMP可以快速、直观、准确地检测单增李斯特菌,是一种适合基层现场应用的检测方法。在此基础上,研制组装的单增李斯特菌快速检测试剂盒,63℃,45min内即可检出单增李斯特菌DNA的扩增产物,其最低检测限为3 CFU/m L,特异性为100%,无假阳性和假阴性出现。采用20份样品,进行批内批间重复性试验、效期稳定性试验等验证,结果表明本试剂盒准确、稳定、重现性好,且LAMP检测中包括前期样品处理、DNA提纯等全部过程仅在2h内便能完成。本课题还在所研究的环介导等温扩增技术基础上,结合横向流动试纸条技术(Lateral flow dipstick,LFD)原理,设计出一种便捷、快速的检测单增李斯特菌的方法。研究过程中,针对单增李斯特菌hly A基因设计4条特异性引物和1条异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记的探针。根据这种方法检测发现,生物素标记LAMP扩增产物可以特异性地与FITC标记的探针杂交,杂交产物经LFD检测。LAMP体系经优化后,其扩增温度为63℃,反应时间50min,从样品处理直到检测完成仅需80min。LAMP-LFD方法可特异性地检出单增李斯特菌,对其它6株常见食源性致病菌的检测结果均显示阴性;对纯细菌培养物的检测灵敏度为3.6 CFU/m L,是利用外引物建立的PCR方法的100倍。总之,用LAMP-LFD方法检测单增李斯特菌,节约环保,流程简单,结果客观明了,适合医院、养殖中心等场所等进行实时监测,值得广泛推广。
[Abstract]:Listeria monocytogenes (Listeria monocytogenes) is a Gram-positive pathogen of zoonosis of human and animal, which can cause Listeria disease in human and many animals.As early as 1990s, it has been listed as one of the four foodborne pathogenic bacteria. In recent years, the infection of Listeria monocytogenes is on the rise in the world.At present, the traditional method of bacterial detection is complex in operation and long in detection period, so it is difficult to meet the need efficiency of rapid detection of food.Therefore, it is necessary to establish a rapid and convenient method for detection of Listeria monocytogenes.In this study, four specific loop-mediated isothermal amplification amplification primers were designed for Listeria monocytogenes (hereinafter referred to as Listeria monocytogenes) gene, and the reaction conditions were optimized by screening primers.A rapid LAMP detection method for Listeria monocytogenes was established.The results showed that the method could specifically detect Listeria monocytogenes with a detection limit of 3 CFU/m L.Amplification products can be determined by agarose gel electrophoresis and color reaction. Lamp can be used to detect Listeria monocytogenes quickly, intuitively and accurately. It is a suitable method for detection of Listeria monocytogenes in the field.On the basis of this, the amplified products of Listeria monocytogenes DNA could be detected within 45 minutes at 63 鈩，

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