鸡传染性支气管炎病毒的分离与鉴定解决方案.doc

目 录 中文摘要 2 Abstract 3 引言 4 1 材料与方法 5 1.1 材料 5 1.1.1 病料 5 1.1.2鸡胚、鸡红细胞 5 1.1.3 主要试剂 6 1.1.4 主要仪器设备 6 1.1.5 引物 6 1.2 方法 7 1.2.1 病料的采集和处理 7 1.2.2 病毒的增殖与纯化 7 1.2.3 病毒的RT-PCR检测 7 1.2.4 鸡胚尿囊液的血凝活性测定 8 1.2.5 鸡胚尿囊液的RT-PCR检测 8 1.2.6 毒株半数感染量（EID50）的测定 8 2 结果与分析 10 2.1 病鸡剖检结果分析 10 2.2 病毒RT-PCR检测结果分析 10 2.3 传毒结果 11 2.4 传代鸡胚尿囊液的血凝检测结果分析 11 2.5 传代鸡胚尿囊液的RT-PCR检测结果 11 2.6 EID50检测结果分析 11 2.6.1 鸡胚胚病变情况 11 2.6.2 EID50的计算结果 12 3 讨论 12 致谢 14 参考文献 15 鸡传染性支气管炎病毒的分离与鉴定 动物医学专业 指导老师 摘要： 2011年4月17日，山东省临沂某大型养鸡厂送检一批疑似感染传染性支气管炎病毒的病（死）鸡，调查得知该鸡群临床上表现咳嗽、甩鼻，食欲不振，精神不佳，脱水，排白色稀粪，爪干、消瘦等症状且死亡率高，现进行病鸡剖检，病变表现为气管出血，“花斑肾”。采集相关病料研磨并反复冻融后，进行RT-PCR检测结果发现IBV阳性，初步诊断该鸡群发生了鸡传染性支气管炎。将病料接种10日龄SPF鸡胚进行盲传，收集各代尿囊液进行血凝试验、RT-PCR检测，同时观察对鸡胚的致病变特征，结果发现血凝试验均呈阴性，RT-PCR结果IBV表现阳性，新城疫（NDV）、禽流感（AIV）等其他外源病毒阴性，鸡胚表现蜷缩、生长阻滞现象，符合IBV的生物学特征，表示成功分离到了1株鸡传染性支气管炎病毒。 关键词：传染性支气管炎病毒；分离鉴定；RT-PCR Infectious Bronchitis Virus Isolation and Identification Veterinary Medicine Tutor Abstract：April 17, 2011, a number of patients (dead) chickens infected with suspecting infectious bronchitis viruse from a large chicken farm for inspection in Linyi, Shandong Province,survey that the clinical manifestations had cough, thrown nose, loss of appetite, of sorts, dehydration, row of white Xifen, claw dry, weight loss and other symptoms ,and high mortality, now dissect chickens, the lesions showed tracheal bleeding, spotted kidney .Acquisition-related disease, repeated freezing and thawing material after grinding, the RT-PCR test was found positive for IBV, the initial diagnosis of the avian infectious bronchitis in chickens was sure preliminarily . The epidemic materials was inoculated to the 10-day-old SPF chick embryo to blind transfer, allantoic fluid collected for the generation of hemagglutination test, RT-PCR test, and observed the characteristics of the embryo-induced lesions,finding that hemagglutination test were negative, the RT –PCR of IBV results showed positive for Newcastle disease (NDV), avian infl