鸡传染性贫血病毒PCR检测试剂盒的研制及应用-预防兽医专业论文.docxVIP

摘要鸡传染性贫血病是由鸡传染性贫血病毒引起雏鸡的再生障碍性贫血和全身性淋巴细胞萎缩 为特征的传染病。本研究建立检测鸡传染性贫血病毒的PER试剂盒，并进行了初步应用。根据鸡传染性贫血病毒(CIAV)Cux-1毒株的基因组序列设计合成了一对扩增VP2基因的 引物，建立了检测CIAV的PCR特异性反应条件并改进了样品预处理方法。用自制Taq DNA 聚合酶(Taq DNA polymerase)替代了商品化酶。用PCR试剂盒检测已知CIAV毒株，均可扩增到特异性条带；而对MDCC—MSB，细胞、 新城疫病毒(NDV)、鸡马立克氏病病毒(MDV)、传染性法氏囊病病毒(IBDV)、传染性支气 管炎病毒(IBV)、减蛋综合症病毒(EDSV)和大肠杆菌等对照病原样品进行PCR扩增，结果 均为阴性，说明此试剂盒特异性很好。用试剂盒检测CIAV阳性样品，结果表明试剂盒的敏感性达l蟾仙。重复性试验结果表明，组装的试剂盒具有很好的重复性。CIAVPCR试剂盒在．20。C条件下，保存期可达10个月。用试剂盒对人工感染SPF鸡体内CIAV的动态分布结果表明，攻毒后第4d印可从不同脏 器中检出CIAv_DNA，至第16d检出率达到高峰(90％)，24d时检出率下降至50％，32d时检出率 又增至近80％，随后检出率再次下降，至60d时检出率仍高达75％。从不同脏器的检出率来看， 胸腺和脾脏最高，其次是肾脏、骨髓，肝脏、白细胞、法氏囊次之．泄殖腔棉拭子检I山率最低。同时用PCR试剂盒和斑点杂交方法对1011份CIAV人工感染和临床样品的检测结果表明， PCR试剂盒检出阳性样本642份，检出率为64％；斑点杂交方法检出阳性样本260份，捡出率 为26％。应用组装的PCR试剂盒和HCT、ELISA、核酸探针斑点杂交方法对中国部分地区的 父母代和商品代鸡群进行了CIAV感染状况的调查，北京地区样品阳性检出率为81％，河北部 分地区样品阳性检出率为67％。结果表明在北京、河北等地CIAV的感染十分普遍。本研究研制的CIAV PCR试剂盒具有经济、快速、准确的优点，为我国鸡传染性贫血痛的 诊断和监测及流行病学调查提供了良好的技术手段。关罐词：鸡传染性贫血病；PCR；试剂盒AbstractChicken infectious anemia(CIA)is a viral disease caused by chicken infectious anemia virus(ClAV)and characterized by aplastic anemia and generalized lymphoid atrophy with concomitant Jmmunosuppressinn．A detection kit based on polymerase chain reaction(PCRl technique for CIAV was developed and primarily applied．The primers for amplification of VP2 gene were,designed based on the genomic sequence of CIAV Cux·1 strain．The PCR conditions were optimized and the pretreatment method for samples was improved．Taq DNA ploymerease prepared in laboratory was used instead of commercial ones tolower the cost．By using the PCR kit，specific DNA framgent from ClAVs could be amplified．whereas no corresponding framgent was found from MDCC·MSBl cellS，Newcastle disease virus(NDVX Mark’s diseasevirus(MDv)，Infectiousbursaldiseasevirus(1BDV)，Infectiousbronchitisvirus(IBV)，Eggdrop syndrome virus(EDSⅥand Escherichia colt as control samples．It was shown that the PCR kit had a good specificity and repeatability．The result indicated that a minimum of l fg CIAV DNA could be