禽弱毒疫苗中鸡传染性贫血病病毒nested-PCR检测方法的建立  

Establishment of Nested-PCR for Detection of Chicken Infectious Anemia Virus in Attenuated Vaccine

作　　者：李晓晗[1] 房立春[1] 李阳[1] 崔治中[1] 常爽[1] 赵鹏[1] 

机构地区：[1]山东农业大学动物医学院,山东泰安271018

出　　处：《中国家禽》2017年第10期18-22,共5页China Poultry

基　　金：农业部财政专项"种禽场主要垂直传播性疾病疫情调查"项目

摘　　要：疫苗中污染鸡传染性贫血病毒(CIAV)是造成CIAV感染的途径之一,而通常疫苗中CIAV污染剂量较低,应用一般的技术难以检测到。为了更灵敏地检测禽弱毒疫苗中CIAV的污染,根据Gen Bank已发表的CIAV基因序列,针对其保守区设计了外用引物和内用引物,通过优化反应体系和反应条件建立了检测CIAV的nested-PCR检测方法。结果显示,所建立的检测方法灵敏度比常规PCR高100倍,该方法可以检测到1 000羽份弱毒疫苗中1 EID_(50)的低剂量CIAV污染,应用该方法从送检的50个疫苗样品中检测出4个样品为CIAV阳性。该方法与禽白血病病毒、禽网状内皮组织增生症病毒以及马立克氏病病毒等的基因均无交叉反应,具有很好的特异性。提示所建立的nested-PCR灵敏度高、特异性强,可用于检测禽弱毒疫苗中CIAV低剂量的污染。Infectious anemia virus （CIAV）is one of the ways to cause CIAV infection,but the low dose of CIAV in vaccine is usually difficult to detect. To detect the contamination of CIAV in attenuated vaccines for poultry, the inner primers and external primers were designed and synthesized based on the published CIAV genome sequence,and a nested-PCR method for detecting CIAV was developed by optimizing the reaction conditions. The results showed that the detection level of nested-PCR was 100-fold higher than that of conventional PCR. One EIDso of 1 000 dosage attenuated vaccines and 4 positive of 50 samples were determined by nested-PCR method. This detection method had good specificity and no cross-reaction with other virus genes,such as avian leukosis virus,reticuloendotheliosis virus, Marek＇s disease virus. These results indicated that the nested-PCR method had high sensitivity and specificity,and could be used for detection of a low dosage CIAV contamination in attenuated vaccines.

关 键 词：CIAV nested—PCR 灵敏性 特异性 污染 

分 类 号：S855.3[农业科学—临床兽医学]