本文選題：綿羊肺炎支原體 + 膠體金免疫層析技術(shù)　； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae,Mo)是一種引起山羊、綿羊、大角羊和小反芻動物傳染性胸膜肺炎的主要致病菌,主要癥狀為咳嗽、喘息、逐漸消瘦、肺間質(zhì)增生等,病程長達(dá)數(shù)月至數(shù)年,四季均可發(fā)病。國內(nèi)首次從四川邊區(qū)萊斯特種羊(從新西蘭、英國引進(jìn))體內(nèi)分離到Mo病原,并在甘肅、寧夏、內(nèi)蒙、河北、新疆等多個省份先后報道了Mo感染,Mo引起的胸膜肺炎蔓延日趨嚴(yán)重,給養(yǎng)羊業(yè)造成巨大的經(jīng)濟(jì)損失。因此,建立一種快速、高敏的檢測方法對該病的快速篩查和有效預(yù)防具有重要意義。本文從血清學(xué)和病原分子生物學(xué)兩方面入手,探究了以側(cè)向?qū)游鰹榛A(chǔ)的Mo快速檢測方法。本研究表達(dá)并純化了綿羊肺炎支原體的infA、Met、MO-1、MO-2四種原核表達(dá)蛋白,培養(yǎng)綿羊肺炎支原體后分別提取全菌蛋白和膜蛋白。以所有蛋白分別作為檢測抗原組裝捕獲法膠體金試紙條:膠體金標(biāo)記金黃色葡萄球菌A蛋白(SPA),檢測抗原包被檢測線(T線),雞抗SPA抗體包被質(zhì)控線(C線)。以infA、Met、MO-1、MO-2蛋白分別作為檢測抗原組裝雙抗原夾心法膠體金試紙條:膠體金標(biāo)記檢測抗原,同時檢測抗原包被T線。血清樣本加于試紙條上樣墊,10~15 min讀取結(jié)果。試紙條經(jīng)測試有效后篩選檢測抗原,建立Mo的膠體金免疫層析檢測方法。結(jié)果顯示:infA、Met重組蛋白作捕獲法的檢測抗原時,陽性樣品結(jié)果明顯,陰性樣品假陽率較高,約50%。作夾心法的檢測抗原時,陽性反應(yīng)結(jié)果較弱,且有假陽性出現(xiàn);MO-1、MO-2作捕獲法的檢測抗原時,陽性反應(yīng)較弱,且有假陽性,與夾心法結(jié)果一致;Mo的全菌蛋白和膜蛋白作捕獲法的檢測抗原時,特異性較好,前者的檢出率高于后者。因此,本研究以Mo的全菌蛋白作為檢測抗原,初步建立了Mo的捕獲法膠體金免疫層析方法,這種方法對其它病原菌感染的血清均顯陰性,靈敏度為1:100,表明其特異性和靈敏度較好。沒有批內(nèi)差和批間差,重復(fù)性和穩(wěn)定性良好。本文初步建立了Mo膠體金免疫層析方法,進(jìn)一步優(yōu)化后可在基層畜牧獸醫(yī)站推廣使用。本研究結(jié)合降落PCR(Touchdown PCR,TD PCR)和側(cè)向?qū)游黾夹g(shù)(lateral flow assay,LFA)建立了Mo病原分子生物學(xué)檢測方法。兩條特異性引物5'端分別標(biāo)記地高辛和生物素,以提取的基因組為模板進(jìn)行TD PCR;膠體金標(biāo)記抗地高辛抗體,檢測線和質(zhì)控線分別包被鏈霉親和素和羊抗鼠IgG,組裝成LFA方法膠體金核酸層析試紙條。將TD PCR產(chǎn)物滴加于核酸試紙條上樣墊,在15 min內(nèi)讀取檢測結(jié)果,通過對各環(huán)節(jié)的調(diào)整優(yōu)化,建立了檢測Mo核酸的TD PCR-LFA快速檢測方法。結(jié)果顯示:建立的TD PCRLFA方法僅對Mo病原呈陽性,其他病原菌均為陰性,表明該方法特異性良好。其檢測下限為20.6 ng/ml,敏感性較強(qiáng)。TD PCR批間產(chǎn)物、批間試紙條讀取結(jié)果一致,重復(fù)性和穩(wěn)定性較好。本研究建立的Mo檢測方法整合了TD PCR的特異性、敏感性和金標(biāo)條快速、簡單的特性,在2 h內(nèi)可完成檢測。為建立適用于基層和畜牧獸醫(yī)站的病原分子學(xué)診斷方法奠定了基礎(chǔ)。
[Abstract]:Mycoplasma ovipneumoniae (Mo) is a major pathogenic bacteria causing infectious pleuropneumonia in goats, sheep, big horn sheep and small ruminants. The main symptoms are cough, wheezing, gradual emaciation and interstitial lung hyperplasia for several months to several years. It is the first time in Leicester from Sichuan border region. Mo pathogens were isolated from New Zealand and Britain) and Mo infection was reported in many provinces in Gansu, Ningxia, Inner Mongolia, Hebei, Xinjiang and other provinces. The spread of pleuropneumonia caused by Mo was becoming more and more serious, causing huge economic losses to the sheep industry. Therefore, a fast speed, Gao Min detection method for rapid screening and effective prevention of the disease was established. This paper, starting with two aspects of serology and pathogenic molecular biology, explores the rapid detection of Mo based on lateral chromatography. This study expresses and purify the four prokaryotic expression proteins of Mycoplasma sheeppneumoniae, infA, Met, MO-1, MO-2, and extracts whole bacterial protein and membrane protein after the culture of Mycoplasma pneumoniae. All proteins were detected as antigen assembly capture colloid gold strip, colloid gold labeled Staphylococcus aureus A protein (SPA), detection of antigen envelope detection line (T line), chicken anti SPA antibody package on the quality control line (C line). InfA, Met, MO-1, MO-2 protein as antigen assembled double antigen sandwich colloid gold test strip, respectively: colloidal gold mark The antigen was detected and the antigen was detected by T line. The serum samples were added to the sample pad and 10~15 min to read the results. The test paper was tested effectively after testing the antigen and established the colloidal gold immunochromatography detection method of Mo. The results showed that the positive sample results were obvious when infA and Met recombinant protein was used as the capture method to detect the antigen, and the negative samples were false positive. When 50%. was used as a sandwich method to detect antigen, the positive reaction results were weak and there were false positive. When MO-1, MO-2 was used as a capture method to detect antigen, the positive reaction was weak, and there was false positive, which was in accordance with the result of sandwich method. The specificity of the whole bacterial protein and membrane protein of Mo was better than that of the capture method. The detection rate of the former was higher than that of the latter. Therefore, in this study, the total bacterial protein of Mo was used as the antigen to detect the antigen, and a colloidal gold immunochromatography for the capture of Mo was initially established. This method was negative to the serum of other pathogenic bacteria, and the sensitivity was 1:100. It showed that the specificity and sensitivity were good. There was no difference in batch and batch difference, and the reproducibility and stability were good. This paper was preliminary. The Mo colloidal gold immunochromatography (GI) was established, which could be further optimized at the grass-roots animal husbandry and veterinary station. This study combined with the landing PCR (Touchdown PCR, TD PCR) and lateral chromatography (lateral flow assay, LFA) to establish a Mo molecular biological detection method. Two specific primers were labeled with digoxin and biotin, respectively. The genome was taken as a template for TD PCR, and the colloidal gold was labeled with digoxin antibody. The detection line and the quality control line were packed by streptomycin and Sheep anti mouse IgG, respectively, and assembled into a LFA colloid gold nucleic acid test paper. The TD PCR products were added to the sample pad of nucleic acid test paper, and the test results were read in 15 min, and the adjustment of each link was optimized. The rapid detection method of TD PCR-LFA for detecting Mo nucleic acid was established. The results showed that the established TD PCRLFA method was only positive for Mo pathogen and the other pathogenic bacteria were negative, which showed that the method had good specificity. The detection limit of the method was 20.6 ng/ml, the sensitivity of.TD PCR inter batch products was stronger, the reading results of the batch test paper were consistent, repeatability and stability were better. Good. The Mo detection method established in this study integrates the specificity of the TD PCR, the sensitivity and the rapid and simple features of the gold bar, and can be detected within 2 h. It lays the foundation for the establishment of a diagnostic method for the pathogen of grass roots and animal husbandry and veterinary medicine.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.26

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