六种病原微生物微量快速现场检测技术的建立及检测箱组研制
发布时间：2018-10-26 17:23
【摘要】：如今我国经济的发展已逐步与世界经济接轨,经济方面的快速崛起给国家和人民带来便利的同时,还存在一系列问题,生物安全问题作为其中之一,其影响力不容忽视。近些年来,口蹄疫、非洲猪瘟、猪瘟、新城疫、布鲁氏菌、大肠杆菌O157:H7等感染在世界范围内不断发生,对公共卫生安全构成了巨大威胁,也产生了巨大危害。目前,国内外对以上六种病原的检测主要依靠血清学诊断技术,存在的问题是所用的诊断方法特异性、敏感性较低。病原学诊断方法研究较少,方法参差不齐,实用的方法少,可用于现场快速检测的方法更少。本研究针对上述问题进行检测方法的优化及评价,选择可应用于现场检测的仪器组装并研制检测箱组,包括样品处理箱、核酸快速检测箱和免疫学快速检测箱,可以完成PCR、ELISA、胶体金等多种检测方法,为今后进行疫病的现场检测提供快速、方便、有效的技术手段。首先,针对六种不同的病原微生物,查阅相关中外文献,选用具有代表性的引物进行筛选,选用三种不同的Taq酶:康为世纪Es Taq MasterMix酶、Ahram High-speed酶、Takara ExTaq酶。由于试验用到的仪器均为微型化仪器,程序设置及机器参数等与常规实验室仪器不同,进行条件摸索十分必要。将实验室保存的羊布鲁氏菌M5、牛布鲁氏菌104M、猪布鲁氏菌S2、大肠杆菌O157:H7、大肠杆菌、金黄色葡萄球菌、小肠耶尔森氏菌、单增李斯特菌、鲍氏不动杆菌、猪链球菌2型、沙门氏菌甘油菌复苏增菌计数,将新城疫、口蹄疫、猪瘟进行细胞培养扩增后测定滴度,将非洲猪瘟质粒转化后扩增。其次,将本试验检测用到的口蹄疫、猪瘟、新城疫三种扩增培养后的病毒,提取相关的RNA进行反转录RT-PCR实验方法的建立;非洲猪瘟提取质粒后进行PCR方法建立;布鲁氏菌、大肠杆菌O157:H7两种细菌进行扩增培养,提取相关的DNA进行PCR实验方法的建立。建立的方法从特异性、敏感性和重复性三个方面进行评价。建立PCR方法之后,采用该方法扩增相应的基因序列进行琼脂糖凝胶电泳,利用凝胶成像系统进行结果的初步判定。将通过初步判定的目的片段进行胶回收并连接pMD-18T载体进行序列测定,测序结果相似性均为99%以上。最后,购买病原检测ELISA试剂盒,对大肠杆菌O157:H7、猪瘟人工污染样品进行检测,并以PCR检测结果为“金标准”,对应用检测箱组完成的ELISA结果进行简要评价,结果显示ELISA试剂盒能够成功检测出相应的病原,具有良好的效果。
[Abstract]:Nowadays, the economic development of our country has been in line with the world economy step by step. The rapid rise of economy has brought convenience to the country and the people, at the same time, there are still a series of problems, the biosafety problem is one of them, its influence can not be ignored. In recent years, foot-and-mouth disease, African hog fever, Newcastle disease, brucella, Escherichia coli O157:H7 and other infections in the world continue to occur, which has posed a great threat to public health and safety, but also caused great harm. At present, the detection of the six pathogens at home and abroad mainly depends on serological diagnostic techniques, the problem is the specificity of the diagnostic methods used, the sensitivity is low. The methods of etiological diagnosis are less studied, the methods are not uniform, the practical methods are few, and the methods that can be used for field rapid detection are even less. Aiming at the above problems, this study optimizes and evaluates the detection method, selects the instruments that can be used in the field detection and develops the test box group, including sample processing box, nucleic acid rapid detection box and immunological rapid detection box, which can complete PCR,. ELISA, colloidal gold and other detection methods provide rapid, convenient and effective technical means for the field detection of epidemic disease in the future. Firstly, according to six different pathogenic microorganisms, the relevant literatures were reviewed, and the representative primers were selected for screening. Three different Taq enzymes were selected: Kangwei Es Taq MasterMix enzyme and Ahram High-speed enzyme, Takara ExTaq enzyme. Because the instruments used in the test are all miniaturized instruments, the program setting and machine parameters are different from the conventional laboratory instruments, so it is necessary to explore the conditions. Laboratory preservation of Brucella sheep M5, Brucella bovis 104M, Brucella suis S2, Escherichia coli O157: H7, Escherichia coli, Staphylococcus aureus, Yersinia small intestine, Listeria monocytogenes, Acinetobacter baumannii, Streptococcus suis type 2, Salmonella glycerol resuscitation increased bacteria count, Newcastle disease, foot-and-mouth disease, hog fever were cultured and amplified, the titer was determined after cell culture, and the plasmids of African swine fever were transformed and amplified. Secondly, three kinds of viruses, including foot-and-mouth disease (FMD), hog fever (CSF) and Newcastle disease (NDV), were detected in this experiment, and the RNA was extracted to establish the reverse transcription RT-PCR method, and the PCR method was established after the plasmid was extracted from African CSFV. Brucella and Escherichia coli O157:H7 were amplified and cultured, and the relative DNA was extracted to establish the PCR method. The established method was evaluated from three aspects: specificity, sensitivity and repeatability. After the establishment of PCR method, the corresponding gene sequence was amplified by agarose gel electrophoresis, and the results were preliminarily determined by gel imaging system. The target fragment was recovered by gel and sequenced by ligation of pMD-18T vector. The results showed that the similarity of the sequencing results was more than 99%. Finally, ELISA kit was purchased for detection of Escherichia coli O157: H7 and swine fever artificially contaminated samples. The results of PCR detection were taken as the "gold standard" to evaluate the ELISA results of the application test box group. The results showed that the ELISA kit could successfully detect the corresponding pathogens and had a good effect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.6
本文编号：2296445
[Abstract]:Nowadays, the economic development of our country has been in line with the world economy step by step. The rapid rise of economy has brought convenience to the country and the people, at the same time, there are still a series of problems, the biosafety problem is one of them, its influence can not be ignored. In recent years, foot-and-mouth disease, African hog fever, Newcastle disease, brucella, Escherichia coli O157:H7 and other infections in the world continue to occur, which has posed a great threat to public health and safety, but also caused great harm. At present, the detection of the six pathogens at home and abroad mainly depends on serological diagnostic techniques, the problem is the specificity of the diagnostic methods used, the sensitivity is low. The methods of etiological diagnosis are less studied, the methods are not uniform, the practical methods are few, and the methods that can be used for field rapid detection are even less. Aiming at the above problems, this study optimizes and evaluates the detection method, selects the instruments that can be used in the field detection and develops the test box group, including sample processing box, nucleic acid rapid detection box and immunological rapid detection box, which can complete PCR,. ELISA, colloidal gold and other detection methods provide rapid, convenient and effective technical means for the field detection of epidemic disease in the future. Firstly, according to six different pathogenic microorganisms, the relevant literatures were reviewed, and the representative primers were selected for screening. Three different Taq enzymes were selected: Kangwei Es Taq MasterMix enzyme and Ahram High-speed enzyme, Takara ExTaq enzyme. Because the instruments used in the test are all miniaturized instruments, the program setting and machine parameters are different from the conventional laboratory instruments, so it is necessary to explore the conditions. Laboratory preservation of Brucella sheep M5, Brucella bovis 104M, Brucella suis S2, Escherichia coli O157: H7, Escherichia coli, Staphylococcus aureus, Yersinia small intestine, Listeria monocytogenes, Acinetobacter baumannii, Streptococcus suis type 2, Salmonella glycerol resuscitation increased bacteria count, Newcastle disease, foot-and-mouth disease, hog fever were cultured and amplified, the titer was determined after cell culture, and the plasmids of African swine fever were transformed and amplified. Secondly, three kinds of viruses, including foot-and-mouth disease (FMD), hog fever (CSF) and Newcastle disease (NDV), were detected in this experiment, and the RNA was extracted to establish the reverse transcription RT-PCR method, and the PCR method was established after the plasmid was extracted from African CSFV. Brucella and Escherichia coli O157:H7 were amplified and cultured, and the relative DNA was extracted to establish the PCR method. The established method was evaluated from three aspects: specificity, sensitivity and repeatability. After the establishment of PCR method, the corresponding gene sequence was amplified by agarose gel electrophoresis, and the results were preliminarily determined by gel imaging system. The target fragment was recovered by gel and sequenced by ligation of pMD-18T vector. The results showed that the similarity of the sequencing results was more than 99%. Finally, ELISA kit was purchased for detection of Escherichia coli O157: H7 and swine fever artificially contaminated samples. The results of PCR detection were taken as the "gold standard" to evaluate the ELISA results of the application test box group. The results showed that the ELISA kit could successfully detect the corresponding pathogens and had a good effect.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.6
【参考文献】
相关期刊论文 前2条
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2 姚斐,沙莎,陈刚,纪伟尚,徐怀恕;间接酶联免疫吸附技术检测活的非可培养状态的大肠杆菌O157:H7[J];青岛海洋大学学报(自然科学版);2001年02期
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