禽弱毒疫苗中REV低剂量污染检测方法的比较  

Comparison of different methods for detection of lose-dose REV contamination in attenuated vaccine

作　　者：任志浩[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,山东泰安271018

出　　处：《中国兽医学报》2017年第6期1041-1045,共5页Chinese Journal of Veterinary Science

摘　　要：使用污染有禽网状内皮组织增生症病毒(REV)的弱毒疫苗是REV感染途径之一,而通常弱毒疫苗中REV的污染剂量较低,难以检测,本研究对不同方法在检测弱毒疫苗中REV低剂量污染进行了比较研究。将定量好的REV以不同剂量分别人为污染一批商品化新城疫病毒(NDV)弱毒疫苗,提取核酸后分别以TaqMan探针荧光定量RT-PCR检测、常规RT-PCR检测以及PCR产物结合核酸斑点杂交检测等不同方法进行检测。结果显示常规RTPCR检测仅可检测到10TCID50/羽份的REV污染,而另外两种方法均检测到1TCID50/羽份的低剂量REV污染。将人为污染1TCID50/羽份和2TCID50/羽份REV的NDV弱毒疫苗各接种10只SPF鸡,定期针对REV抗体及其核酸进行检测,结果显示在免疫污染疫苗后7周内REV抗体全部为阴性,而核酸检测均可以检测到一定比例的阳性,提示通过接种SPF鸡检测弱毒疫苗中REV污染时,仅仅通过抗体判定可能会对一些剂量较低的REV污染造成漏检,通过结合对病原的检测有助于降低漏检率。Application of attenuated vaccines contaminated with reticuloendotheliosis virus（REV） is a route of REV transmission. However,it is difficult to detect it because of the low-dose contami- nation in those vaccines. In this study, a batch of Newcastle disease virus（NDV） attenuated vac- cines were artificially contaminated with REV. RNAs were extracted and three different approa- ches were performed,including TaqMan real-time RT-PCR, routine RT-PCR, and RT-PCR in com- bination with dot-blot hybridization technology. The results showed that extremely low dose of REV as 10-3 TCIDs0 per portion could be detected by real-time RT-PCR or RT-PCR in combina- tion with dot-blot technology,while only 10 TCID50 per portion could be detected by routine RT- PCR. Then, attenuated NDV vaccines contaminated with 1 TCID50 or 2 TCID50 per feather REV were injected with SPF chickens,and REV antibody and viral RNA were detected regularly. The results showed that no REV antibody could be detected untill 7 weeks post-vaccination, while a portion of positive rates was detected by viral RNA detection. It could be concluded that misdetec- tion inspection might occur if it was only determined by REV antibody response when chickens were inoculated vaccines with low-dose REV contamination. A synthetic determination combined with etiological detection would reduce the misdetection rate.

关 键 词：禽网状内皮组织增生症病毒 污染 REAL-TIME RT-PCR 核酸斑点杂交 

分 类 号：S852.65[农业科学—基础兽医学]