鸡传染性贫血病毒荧光定量PCR检测方法的建立及其在疫苗污染检测中的应用  

Development and Application of a Quantitative PCR for Detection of Chicken Infectious Anemia Virus Contamination in Attenuated Vaccines

作　　者：任志浩[1] 房立春[1] 栾怀彪 李阳[1] 王一新[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,泰安271018

出　　处：《畜牧兽医学报》2016年第7期1459-1464,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：农业部财政项目"禽垂直传播性疾病诊断技术研究"

摘　　要：为了更快速而灵敏地检测禽弱毒疫苗中鸡传染性贫血病毒(CIAV)的污染,根据CIAV基因组保守序列设计合成了引物及TaqMan探针,通过优化反应条件建立了快速检测CIAV的实时荧光定量PCR检测方法。结果显示,建立的检测方法灵敏度可达10拷贝·μL^(-1),比常规PCR灵敏度高100倍。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病病毒等病毒基因均无交叉反应,具有很好的特异性;批内重复和批间重复显示变异系数均小于2%,呈现良好的可重复性。在模拟试验中,该方法能够检测到1 000羽份弱毒疫苗中1个EID50的低剂量污染,应用该方法从送检的39种(批)商品化禽用弱毒疫苗中检测到2份为CIAV阳性。上述结果表明,本研究建立的荧光定量PCR检测方法灵敏度高,特异性强,重复性好,可用于检测疫苗中CIAV低剂量的污染。To detect the contamination of Chicken infectious anemia virus（CIAV）in attenuated vaccines for poultry,primers and TaqMan probes were designed and synthesized based on the published CIAV genome sequence,and a real-time quantitative PCR method for detecting the CIAV was established by optimizing the reaction conditions.Results analysis showed that the sensitivity of the qPCR may increase detection to 10copies·μL-1,and the method was approximately100-fold higher than the sensitivity of the conventional PCR.This detection method had good specificity,and showed no cross-reactions with ALV,REV and MDV genes,the CV（coefficient of variation）of intra-assay and inter-assay were less than 2%,and also showed that the method had the characteristics of better repeatability.In our experiment,a low dosage of 1EID50·1 000-1plumes and in 2positive（5.1%）of 39 samples were detected for CIAV detection from commercial poultry vaccines by qPCR method.In this study,suggesting that this method provides a high sensitivity,strong specificity and better repeatability system for detection of a low dosage CIAV contamination in attenuated vaccines.

关 键 词：鸡传染性贫血病毒 实时荧光定量PCR 弱毒疫苗 污染 

分 类 号：S852.659.2[农业科学—基础兽医学]