鸭肝炎病毒PCR-ELISA检测方法的建立  

Development of PCR-ELISA method for detection of duck hepatitis virus

作　　者：耿昕颖 刘艳萍[1] 张福君 王巍[1] 毕彦晖 马红霞[1] 高云航[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林正方农牧股份有限公司,吉林梅河口135000

出　　处：《中国兽医科学》2016年第2期161-166,共6页Chinese Veterinary Science

基　　金：国家现代农业技术体系建设专项(CARS-43);吉林省科技发展计划项目(20111820);国家星火计划项目(2012GA-660003)

摘　　要：为建立检测鸭肝炎病毒(DHV)的PCR-ELISA方法,根据DHV VP1基因序列设计1对分别标记生物素和地高辛的引物,采用固相杂交法,将生物素标记的探针固定在酶标板上,再与地高辛标记的产物进行杂交,并通过反复优化确定了10 mg/m L链霉亲和素包被液、10 g/L BSA封闭液、0.5 pmol/L探针、1∶4 000稀释的酶标二抗及最适杂交时间等最佳反应条件,建立了DHV PCR-ELISA检测方法。该方法与鸭肠炎病毒、鸭细小病毒和鸭疫里氏杆菌等无交叉反应,说明该方法特异性强;敏感性试验结果表明,PCR-ELISA最低能检出0.37 pg/L的DHV RNA,其敏感度比常规PCR高100倍。用该方法对50份雏鸭肝提取的RNA样本进行检测,阳性检出率为82%,较RT-PCR检出率高46%。结果表明,该PCR-ELISA方法具有特异、敏感、安全的特点,从而为鸭病毒性肝炎流行病学研究及临床诊断提供了新的途径。In order to develop a PCR-ELISA for the detection of duck hepatitis virus（DHV）, the PCR primers labeled by biotin and digoxin were designed according to VP1 gene of DHV. The solid-phase hybridization method was used to determine the optimum reaction conditions including streptavidin coating buffer at 10 mg/m L, blocking buffer at 10 g/L BSA, probe at 0.5 pmol/L,and enzyme-labeled second antibody dilution at 1 ∶ 4 000. The specificity test showed that there was no cross reaction with duck enteritis virus,duck parvovirus and Riemerella anatipestifer,respectively.The sensitivity test showed that the detected threshold was 0.37 pg/ L of DHV,and its sensitivity was 100 times higher than the conventional PCR method. This PCR-ELISA was tested on 50 RNA samples from ducks,the HV positive rate was82%,which is higher than that of RT-PCR（36%）：The results indicated that the PCR-ELISA could be a appropriate method with specificity,sensitivity,and safety,thereby it could provide a new tool for epidemiological study and clinical diagnosis of duck viral hepatitis.

关 键 词：鸭肝炎病毒 VP1基因 PCR-ELISA 

分 类 号：S852.659.6[农业科学—基础兽医学]