六種病原微生物微量快速現(xiàn)場檢測技術(shù)的建立及檢測箱組研制
發(fā)布時(shí)間：2018-10-26 17:23
【摘要】：如今我國經(jīng)濟(jì)的發(fā)展已逐步與世界經(jīng)濟(jì)接軌,經(jīng)濟(jì)方面的快速崛起給國家和人民帶來便利的同時(shí),還存在一系列問題,生物安全問題作為其中之一,其影響力不容忽視。近些年來,口蹄疫、非洲豬瘟、豬瘟、新城疫、布魯氏菌、大腸桿菌O157:H7等感染在世界范圍內(nèi)不斷發(fā)生,對(duì)公共衛(wèi)生安全構(gòu)成了巨大威脅,也產(chǎn)生了巨大危害。目前,國內(nèi)外對(duì)以上六種病原的檢測主要依靠血清學(xué)診斷技術(shù),存在的問題是所用的診斷方法特異性、敏感性較低。病原學(xué)診斷方法研究較少,方法參差不齊,實(shí)用的方法少,可用于現(xiàn)場快速檢測的方法更少。本研究針對(duì)上述問題進(jìn)行檢測方法的優(yōu)化及評(píng)價(jià),選擇可應(yīng)用于現(xiàn)場檢測的儀器組裝并研制檢測箱組,包括樣品處理箱、核酸快速檢測箱和免疫學(xué)快速檢測箱,可以完成PCR、ELISA、膠體金等多種檢測方法,為今后進(jìn)行疫病的現(xiàn)場檢測提供快速、方便、有效的技術(shù)手段。首先,針對(duì)六種不同的病原微生物,查閱相關(guān)中外文獻(xiàn),選用具有代表性的引物進(jìn)行篩選,選用三種不同的Taq酶:康為世紀(jì)Es Taq MasterMix酶、Ahram High-speed酶、Takara ExTaq酶。由于試驗(yàn)用到的儀器均為微型化儀器,程序設(shè)置及機(jī)器參數(shù)等與常規(guī)實(shí)驗(yàn)室儀器不同,進(jìn)行條件摸索十分必要。將實(shí)驗(yàn)室保存的羊布魯氏菌M5、牛布魯氏菌104M、豬布魯氏菌S2、大腸桿菌O157:H7、大腸桿菌、金黃色葡萄球菌、小腸耶爾森氏菌、單增李斯特菌、鮑氏不動(dòng)桿菌、豬鏈球菌2型、沙門氏菌甘油菌復(fù)蘇增菌計(jì)數(shù),將新城疫、口蹄疫、豬瘟進(jìn)行細(xì)胞培養(yǎng)擴(kuò)增后測定滴度,將非洲豬瘟質(zhì)粒轉(zhuǎn)化后擴(kuò)增。其次,將本試驗(yàn)檢測用到的口蹄疫、豬瘟、新城疫三種擴(kuò)增培養(yǎng)后的病毒,提取相關(guān)的RNA進(jìn)行反轉(zhuǎn)錄RT-PCR實(shí)驗(yàn)方法的建立;非洲豬瘟提取質(zhì)粒后進(jìn)行PCR方法建立;布魯氏菌、大腸桿菌O157:H7兩種細(xì)菌進(jìn)行擴(kuò)增培養(yǎng),提取相關(guān)的DNA進(jìn)行PCR實(shí)驗(yàn)方法的建立。建立的方法從特異性、敏感性和重復(fù)性三個(gè)方面進(jìn)行評(píng)價(jià)。建立PCR方法之后,采用該方法擴(kuò)增相應(yīng)的基因序列進(jìn)行瓊脂糖凝膠電泳,利用凝膠成像系統(tǒng)進(jìn)行結(jié)果的初步判定。將通過初步判定的目的片段進(jìn)行膠回收并連接pMD-18T載體進(jìn)行序列測定,測序結(jié)果相似性均為99%以上。最后,購買病原檢測ELISA試劑盒,對(duì)大腸桿菌O157:H7、豬瘟人工污染樣品進(jìn)行檢測,并以PCR檢測結(jié)果為“金標(biāo)準(zhǔn)”,對(duì)應(yīng)用檢測箱組完成的ELISA結(jié)果進(jìn)行簡要評(píng)價(jià),結(jié)果顯示ELISA試劑盒能夠成功檢測出相應(yīng)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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.6
本文編號(hào)：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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.6
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 常華;花群義;段綱;項(xiàng)勛;曾昭文;;非洲豬瘟的研究進(jìn)展[J];中國畜牧獸醫(yī);2007年01期
2 姚斐,沙莎,陳剛,紀(jì)偉尚,徐懷恕;間接酶聯(lián)免疫吸附技術(shù)檢測活的非可培養(yǎng)狀態(tài)的大腸桿菌O157:H7[J];青島海洋大學(xué)學(xué)報(bào)(自然科學(xué)版);2001年02期
，本文編號(hào)：2296445
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