鸡传染性贫血病毒的环介导等温扩增检测系统的建立与应用

发布时间：2018-05-13 14:03

  本文选题：鸡传染性贫血病毒 + 环介导等温扩增　； 参考：《南阳师范学院》2017年硕士论文

【摘要】：鸡传染性贫血病(Chicken Infectious Anemia,CIA)是由鸡传染性贫血病毒(Chicken Infectious Anemia Virus,CIAV)所引起的鸡造血器官和淋巴组织损害而导致的一种再生障碍性贫血性疾病,也是在全世界的养禽生产中是较为多发的一种免疫抑制性疾病。临床上经常合并、继发和加重病毒、细菌和真菌性感染,产生混合感染,严重影响雏鸡的生长发育。该病已成为当前严重危害我国养鸡业的传染病之一,应引起高度重视,而建立准确快速的诊断方法是防控该病的前提,具有重要的临床推广意义。环介导等温扩增检测法(loop-mediated isothermal amplification,LAMP)是一种新兴的核酸扩增检测方法,以此检测方法为基础为基础,通过对该方法的优化,建立了一套广泛应用的CIAV快速检测系统,使CIAV的检测更方便、快速,成本更低,有利于鸡传染性贫血病的大规模大范围检测。主要研究结果如下：1.采用羟基溴酚蓝作为LAMP反应的染料,反应结束后通过肉眼观察颜色变化即可对样品的阴性和阳性进行判断,可视性强,操作简便。2.利用Blast、Meg Align和DNA Star对CIAV的VP2基因的相对保守段设计了4条特异性引物。以LAMP检测方法为基础,通过不断的探索和条件优化,建立灵敏、特异的环介导等温扩增反应体系。优化体系如下：25μL反应体系,加入羟基溴酚蓝(HNB)染料至终浓度为240 μmol/L时,LAMP检测结果较好；通过调节Mg离子的浓度发现,在25μL体系中Mg离子浓度为0.4 mmol/L时,显色结果较好；对温度进行优化,发现基于本研究设计的LAMP引物可实现宽温型检测,在55～65℃之间均能有效检测,有利于使用水浴锅等非精确温控设备进行检测,结合琼脂糖凝胶结果,最佳反应温度确定为61℃；当对LAMP进行20～70min梯度反应时,50 min即可快速检测出CIAV;对内引物浓度进行优化时发现,在内引物浓度为0.6时就可以得到良好结果。通过以上条件的优化,建立了最优的CIAV的LAMP检测反应体系。3.构建CIAV克隆载体,将CIAV重组质粒梯度稀释,验证LAMP检测方法的灵敏性,反应产物分别肉眼和琼脂糖凝胶检测,并与常规PCR反应比较,结果表明LAMP灵敏度是普通PCR的10倍。分别提取禽腺病毒(FAVI)1群、减蛋下降综合征病毒(EDS)、鸡传染性喉气管炎病毒(ILT)、鸡马立克氏病病毒(MD)、鸡毒支原体的DNA (MG)、CIAV阳性样品与正常鸡组织DNA,用LAMP进行检测。结果表明,该检测方法只能检测出CIAV。当对20份临床样品进行LAMP检测时,LAMP与普通PCR的阳性检出率符合度高达100%,显示LAMP检测系统高效准确。环介导等温扩增为开发出检测CIAV的试剂盒奠定了基础,具有一定的应用推广价值。
[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，

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