半套式RT-PCR检测动物狂犬病病毒的研究  

Study on Detection of Animal Rabies Virus by Semi-nested RT-PCR

作　　者：袁颖烁[1,2] 赵敬慧[2] 宋菲菲[1,2] 刘晔[2] 张守峰[2] 张乐萃[1] 扈荣良[2] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医杂志》2013年第5期13-16,共4页Chinese Journal of Veterinary Medicine

基　　金：国家公益性行业(农业)科研专项经费资助(201203056)

摘　　要：基于我国流行的狂犬病病毒(Rabies Virus,RV)均属基因I型,本研究根据GenBank公布的31株RV N基因全序列,选取保守区域设计2对引物,建立了一种检测RV的半套式聚合酶链式反应(Semi-nested RT-PCR),并对引物的灵敏性、敏感性和特异性进行检测。将该方法检测的36份新鲜脑组织样品,与荧光抗体染色法(FAT)进行比较,并将PCR结果为阳性的新鲜样品室温下放置腐败后进行检测。结果显示,该方法灵敏度检测可达到0.01TCID50/mL。对检测呈阳性的病料进行序列分析,全部为狂犬病病毒;检测犬细小病毒(CPV)、犬瘟热病毒(CDV)、犬腺病病毒(CAV)、伪狂犬病病毒(PRV)等非狂犬病病毒,结果均为阴性,表明该引物特异性较高。该方法与FAT检测结果完全一致,并能从腐败样品中检出RV,更适用于腐败样品的检测,具有快速、准确的特点,可广泛应用于广大基层狂犬病的诊断及试验研究。Based on the gene type which the epidemic strains of Rabies Virus in China belong to is the type I, a semi-nested polymerase chain was established in order to detect rabies virus, using a couple of primers designed by selecting ＇the conserved regions of the nucleoprotein gene of 31 strains RV in Gen- Bank. In this study, the sensitivity and specificity of primers were detected. 36 samples of fresh brain tis- sues were tested and compared to rabies diagnostic gold standard-fluorescent antibody staining （FAT）. The fresh positive samples placed at room temperature resulting in corruption were detected using this method. The results showed that the sensitivity can reach up to 0.01TCID50/mL. All positive samples were RV by sequence analysisl the non-rabies viruses detected such as canine parvovirus （CPV）, canine distemper virus （CDV）, canine adenoviral virus （CAV）, pseudo-rabies virus （PRV） were negative, indi- cating that the specificity of this method is relatively high. This method is completely consistent with the results of the FAT, and more suitable for the detection of rabies virus in corrupted samples. The results suggest that the protocol is not only fast but accurate, and can be widely used for the diagnosis of animal rabies in the numerous grassroot units and experimental study.

关 键 词：狂犬病病毒 半套式RT-PCR 诊断 

分 类 号：S852.659.4[农业科学—基础兽医学]