AIV、ALV、IBV、IBDV四重RT-PCR检测技术研究
发布时间：2019-03-25 08:46
【摘要】：禽流感病毒(Avianinfluenzavirus, AIV).禽白血病病毒(Avianleukosisvirus,ALV)、禽传染性支气管炎病病毒(Infectiousbronchitisvirus, IBV)、鸡传染性法氏囊病病毒(Infectiousbursaldiseasevirus, IBDV)是四种严重危害家禽养殖业的禽类传染病重要病原。建立AIV. ALV. IBV和IBDV特异灵敏的检测技术,对于早期发现传染源,及尽早采取措施消除传染源,切断疾病传播途径,降低疾病危害具有重要意义。本研究根据ALV病毒M基因的保守序列,利用Primer Premier 5.0设计ALV简并引物,参照文献选择AIV、IBV、IBDV的引物,以从AIV、ALV、IBV和IBDV的细胞培养液中抽提的病毒RNA作为模板,分别RT-PCR扩增了他们各自特异的基因片段,成功构建阳性对照质粒pMD-18T-AIV、pMD-18T-ALV、pMD-18T-IBV和pMD-18T-IBDV。通过退火温度、引物比例、引物浓度、dNTP浓度、Mg2+浓度和PCR buffer浓度等条件的优化,确定AIV、ALV、IBV和IBD V四重PCR最佳反应条件为：AIV与IBV引物浓度为0.125μM, ALV与IBDV引物浓度为0.1875μM, PCR Buffe浓度为1.5×, Taq DNA聚合酶用量为0.075U/μL, Mg2+浓度为2.5mM, dNTP浓度为200μM。最佳退火温度为52℃。以10倍系列稀释的pMD-18T-AIV、pMD-18T-ALV、pMD-18T-IBV、作为模板扩增结果显示,四重PCR检测ApMD-18T-IBDV的灵敏度分别为102拷贝、103拷贝、102拷贝、103拷贝。以10倍系列稀释的AIV、IV、ALV、IBV和IBDV和IBDV作为模板扩增结果显示,四重RT-PCR检测AIV、ALV、IBVALV、IBV和IBDV的灵敏度分别为四重PCR扩增0.0001TCID50、100fg、0.00001TCID50和0.001TCID50。或其中2、3和4种质粒组合时,均可相应产生1、2、3或4条特异性条带；而检pMD-18T-AIV.pMD-18T-ALV.pMD-18T-IBV和pMD-18T-IBDV测其它几种禽类病毒基因克隆质粒时,均无特异性条APV、EDSV、MDV和NDV带产生。四重扩增RT-PCR核酸或其中2、3和4种核酸AIV.ALV、IBV和IBDV组合时,也均相应产生1、2、3和4条特异性条带；而检测其它几种禽类病毒APV、和NDV核酸时,均无特异性条带产生。表明本研究建立的EDSV、MDV四重RT-PCR具有高度特异性,一次扩增可检测可以检测出AIV、ALV、 IBV和IBDV中任1种,或其中2、3和4种病毒的混合。AIV、ALV、 IBV和IBDV将细胞培养的单独或其中2、3或4种病毒混合添加AIV、ALV、IBV、IBDV到经单一PCR检测阴性的禽类粪便制备人工阳性样品,四重RT-PCR扩增结果显示,该方法可准确检测出四种病毒中之1种,或其中2、3和4种病毒混合的人工阳性样品。表明本研究建立的AIV、ALV、IBV和IBDV四重RT-PCR不仅可以用于实验室培养病毒的检测,也适用于禽类组织和排泄物材料等临床样品中AIV、ALV、IBV和IBDV的检测。在武汉市虎泉集贸市场采集鸡、鸭和鸽子粪便样品各10份,在湖北省武汉市、大冶市采集六科7属9种野生鸟类67只,分别提取病毒RNA四重RT-PCR检测,结果在鸡、鸭和鸽子粪便样品中未检测到AIV、ALV、IBV和BDV,67只野生鸟类组织样品中,自大冶市采集的6只斑鸠及武汉市采集的1只领雀嘴鹎IBDV检测阳性,AIV、ALV和IBV检测阴性。检测结果为IBDV的分子流行病学及分子溯源提供了依据。
[Abstract]:Avian influenza virus (Avianinfluenzavirus, AIV). Avian Leukosis virus (Avianleukosisvirus,ALV), Avian Infectious bronchitis virus (Infectiousbronchitisvirus, IBV), Chicken Infectious bursal Disease virus (Infectiousbursaldiseasevirus, IBDV) is one of the four important pathogens of avian infectious diseases which seriously harm the poultry industry. Establish AIV. ALV. The specific and sensitive detection of IBV and IBDV is of great significance for early detection of infection source, and early measures to eliminate infection source, cut off the transmission route of disease and reduce the harm of disease. In this study, according to the conserved sequence of M gene of ALV virus, the degenerate ALV primers were designed by Primer Premier 5.0.According to the literature, the primers of AIV,IBV,IBDV were selected and the virus RNA extracted from the cell culture medium of AIV,ALV,IBV and IBDV was used as the template. Their specific gene fragments were amplified by RT-PCR respectively, and the positive control plasmids pMD-18T-AIV,pMD-18T-ALV,pMD-18T-IBV and pMD-18T-IBDV. were constructed successfully. Through the optimization of annealing temperature, primer ratio, primer concentration, dNTP concentration, Mg2 concentration and PCR buffer concentration, the optimum reaction conditions of AIV,ALV,IBV and IBD V quadruple PCR were determined as follows: the concentration of AIV and IBV primer was 0.125 渭 M, The primer concentration of ALV and IBDV was 0.1875 渭 M, the concentration of, PCR Buffe was 1.5 脳, Taq DNA polymerase dosage was 0.075U/ 渭 L, the concentration of Mg2 was 2.5 mm, the concentration of dNTP was 200 渭 M. The optimum annealing temperature is 52 鈩，
本文编号：2446824
[Abstract]:Avian influenza virus (Avianinfluenzavirus, AIV). Avian Leukosis virus (Avianleukosisvirus,ALV), Avian Infectious bronchitis virus (Infectiousbronchitisvirus, IBV), Chicken Infectious bursal Disease virus (Infectiousbursaldiseasevirus, IBDV) is one of the four important pathogens of avian infectious diseases which seriously harm the poultry industry. Establish AIV. ALV. The specific and sensitive detection of IBV and IBDV is of great significance for early detection of infection source, and early measures to eliminate infection source, cut off the transmission route of disease and reduce the harm of disease. In this study, according to the conserved sequence of M gene of ALV virus, the degenerate ALV primers were designed by Primer Premier 5.0.According to the literature, the primers of AIV,IBV,IBDV were selected and the virus RNA extracted from the cell culture medium of AIV,ALV,IBV and IBDV was used as the template. Their specific gene fragments were amplified by RT-PCR respectively, and the positive control plasmids pMD-18T-AIV,pMD-18T-ALV,pMD-18T-IBV and pMD-18T-IBDV. were constructed successfully. Through the optimization of annealing temperature, primer ratio, primer concentration, dNTP concentration, Mg2 concentration and PCR buffer concentration, the optimum reaction conditions of AIV,ALV,IBV and IBD V quadruple PCR were determined as follows: the concentration of AIV and IBV primer was 0.125 渭 M, The primer concentration of ALV and IBDV was 0.1875 渭 M, the concentration of, PCR Buffe was 1.5 脳, Taq DNA polymerase dosage was 0.075U/ 渭 L, the concentration of Mg2 was 2.5 mm, the concentration of dNTP was 200 渭 M. The optimum annealing temperature is 52 鈩，
本文编号：2446824
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2446824.html
