本文選題：雞傳染性貧血病毒 + 環(huán)介導(dǎo)等溫?cái)U(kuò)增�。� 參考：《南陽(yáng)師范學(xué)院》2017年碩士論文

【摘要】：雞傳染性貧血病(Chicken Infectious Anemia,CIA)是由雞傳染性貧血病毒(Chicken Infectious Anemia Virus,CIAV)所引起的雞造血器官和淋巴組織損害而導(dǎo)致的一種再生障礙性貧血性疾病,也是在全世界的養(yǎng)禽生產(chǎn)中是較為多發(fā)的一種免疫抑制性疾病。臨床上經(jīng)常合并、繼發(fā)和加重病毒、細(xì)菌和真菌性感染,產(chǎn)生混合感染,嚴(yán)重影響雛雞的生長(zhǎng)發(fā)育。該病已成為當(dāng)前嚴(yán)重危害我國(guó)養(yǎng)雞業(yè)的傳染病之一,應(yīng)引起高度重視,而建立準(zhǔn)確快速的診斷方法是防控該病的前提,具有重要的臨床推廣意義。環(huán)介導(dǎo)等溫?cái)U(kuò)增檢測(cè)法(loop-mediated isothermal amplification,LAMP)是一種新興的核酸擴(kuò)增檢測(cè)方法,以此檢測(cè)方法為基礎(chǔ)為基礎(chǔ),通過(guò)對(duì)該方法的優(yōu)化,建立了一套廣泛應(yīng)用的CIAV快速檢測(cè)系統(tǒng),使CIAV的檢測(cè)更方便、快速,成本更低,有利于雞傳染性貧血病的大規(guī)模大范圍檢測(cè)。主要研究結(jié)果如下：1.采用羥基溴酚藍(lán)作為L(zhǎng)AMP反應(yīng)的染料,反應(yīng)結(jié)束后通過(guò)肉眼觀察顏色變化即可對(duì)樣品的陰性和陽(yáng)性進(jìn)行判斷,可視性強(qiáng),操作簡(jiǎn)便。2.利用Blast、Meg Align和DNA Star對(duì)CIAV的VP2基因的相對(duì)保守段設(shè)計(jì)了4條特異性引物。以LAMP檢測(cè)方法為基礎(chǔ),通過(guò)不斷的探索和條件優(yōu)化,建立靈敏、特異的環(huán)介導(dǎo)等溫?cái)U(kuò)增反應(yīng)體系。優(yōu)化體系如下：25μL反應(yīng)體系,加入羥基溴酚藍(lán)(HNB)染料至終濃度為240 μmol/L時(shí),LAMP檢測(cè)結(jié)果較好；通過(guò)調(diào)節(jié)Mg離子的濃度發(fā)現(xiàn),在25μL體系中Mg離子濃度為0.4 mmol/L時(shí),顯色結(jié)果較好；對(duì)溫度進(jìn)行優(yōu)化,發(fā)現(xiàn)基于本研究設(shè)計(jì)的LAMP引物可實(shí)現(xiàn)寬溫型檢測(cè),在55～65℃之間均能有效檢測(cè),有利于使用水浴鍋等非精確溫控設(shè)備進(jìn)行檢測(cè),結(jié)合瓊脂糖凝膠結(jié)果,最佳反應(yīng)溫度確定為61℃；當(dāng)對(duì)LAMP進(jìn)行20～70min梯度反應(yīng)時(shí),50 min即可快速檢測(cè)出CIAV;對(duì)內(nèi)引物濃度進(jìn)行優(yōu)化時(shí)發(fā)現(xiàn),在內(nèi)引物濃度為0.6時(shí)就可以得到良好結(jié)果。通過(guò)以上條件的優(yōu)化,建立了最優(yōu)的CIAV的LAMP檢測(cè)反應(yīng)體系。3.構(gòu)建CIAV克隆載體,將CIAV重組質(zhì)粒梯度稀釋,驗(yàn)證LAMP檢測(cè)方法的靈敏性,反應(yīng)產(chǎn)物分別肉眼和瓊脂糖凝膠檢測(cè),并與常規(guī)PCR反應(yīng)比較,結(jié)果表明LAMP靈敏度是普通PCR的10倍。分別提取禽腺病毒(FAVI)1群、減蛋下降綜合征病毒(EDS)、雞傳染性喉氣管炎病毒(ILT)、雞馬立克氏病病毒(MD)、雞毒支原體的DNA (MG)、CIAV陽(yáng)性樣品與正常雞組織DNA,用LAMP進(jìn)行檢測(cè)。結(jié)果表明,該檢測(cè)方法只能檢測(cè)出CIAV。當(dāng)對(duì)20份臨床樣品進(jìn)行LAMP檢測(cè)時(shí),LAMP與普通PCR的陽(yáng)性檢出率符合度高達(dá)100%,顯示LAMP檢測(cè)系統(tǒng)高效準(zhǔn)確。環(huán)介導(dǎo)等溫?cái)U(kuò)增為開發(fā)出檢測(cè)CIAV的試劑盒奠定了基礎(chǔ),具有一定的應(yīng)用推廣價(jià)值。
[Abstract]:Chicken Infectious AnemiaCIAA is a disease of aplastic anemia caused by the damage of hematopoietic organs and lymphoid tissues caused by Chicken Infectious Anemia virus. It is also an immunosuppressive disease in poultry production all over the world. Clinical infection is often combined, secondary and aggravated by viruses, bacteria and fungal infections, resulting in mixed infection, which seriously affects the growth and development of chicks. The disease has become one of the infectious diseases which seriously endangers the chicken industry in our country at present, which should be paid great attention to, and the establishment of accurate and rapid diagnosis method is the premise of prevention and control of the disease, which has important clinical significance. Loop-mediated isothermal amplification method (lap) is a new method for nucleic acid amplification. Based on this method, a set of widely used CIAV rapid detection system has been established by optimizing the method. The detection of CIAV is more convenient, rapid and low cost, which is beneficial to the large-scale detection of chicken infectious anemia. The main results are as follows: 1. Hydroxybromophenol blue was used as the dye of LAMP reaction. After the reaction, the negative and positive samples could be judged by observing the color changes with naked eye. Four specific primers were designed using Blastnmeg Align and DNA Star for the relative conserved segment of VP2 gene of CIAV. Based on LAMP detection method, a sensitive and specific system of cyclized isothermal amplification was established by continuous exploration and optimization of conditions. The optimized reaction system is as follows: 1: 25 渭 L reaction system. When the final concentration is 240 渭 mol/L, the detection result of lamp is better, and when mg ion concentration is 0. 4 mmol/L in 25 渭 L system, the result of coloration is better. By optimizing the temperature, it is found that the LAMP primer designed in this study can realize the wide temperature type detection, and can be effectively detected between 55 鈩，

本文編號(hào)：1883443