摘要
为建立流行性出血病病毒(EHDV)病原学检测方法,本试验用纯化处理的4种血清型病毒(EHDV-2、5、6和8)混合免疫兔和豚鼠,制备高免血清。以兔抗EHDV为捕获抗体,豚鼠抗EHDV为检测抗体,羊抗豚鼠IgG-HRP为酶标抗体,建立了EHDV抗原捕获ELISA(antigen capture ELISA,AC-ELISA)方法。通过反应条件优化,获得最佳工作条件:5%马血清为稀释液,包被抗体1∶15 000稀释,检测抗体1∶20 000稀释,酶标抗体1∶15 000稀释。通过检测60份阴性样品确定临界值,P/N值≥2为阳性、P/N值<1.5为阴性;特异性试验表明该方法仅能检测出各血清型EHDV、蓝舌病病毒(BTV)、赤羽病病毒(AKAV)及中山病毒(CHUV)无交叉反应;敏感性试验表明该方法可以检出102.47±0.07 TCID50的病毒;批内和批间变异系数均<10%;用RT-PCR与该方法同时对70份参考样品进行检测,符合率为92.86%。表明本试验建立的AC-ELISA方法为EHDV抗原检测提供了一种可靠实用的技术手段。
To establish an assay for epizootic haemorrhagic disease virus(EHDV)detection,the anti-serum against EHDV were prepared with the purified EHDV-2,5,6and 8replicated in BHK-21 cell to immune the rabbit and guinea pig respectively,and then the antigen capture ELISA(ACELISA)was developed with the rabbit anti-serum as coating antibody for antigen capture and with the guinea pig anti-serum for sandwich binding antigen follow by IgG-HRP detection.The optimum conditions were achieved:5% horse serum as diluents,capture antisera was diluted for 1∶15 000,detecting antibody was diluted 1∶20 000 and the goat to Gpig IgG-HRP was diluted for1∶15 000.Using P/N value as the criteria by setting positive and negative control,serum sample with P/N ration more than or equal to 2.0were considered as positive,those less than 1.5as negative,and the rest was suspicious.The AC-ELISA was high specificity and sensitive for EHDV detection with a limit detection of 102.47±0.07 TCID50 and no cross-reaction was found in bluetongue virus,Akabane virus and Chuzan disease virus.The intra-assay and inter-assay variation coefficient were both less 10%,and the coincidence rate with RT-PCR was 92.86%.These results demonstrated the AC-ELISA was sensitive,specific and accurate detection technology for EHDV.
作者
杨振兴
朱建波
肖雷
高林
苗海生
何于雯
孟锦昕
杨恒
李华春
YANG Zhen-xing;ZHU Jian-bo;XIAO Lei;GAO Lin;MIAO Hai-sheng;HE Yu-wen;MENG Jin-xin;YANG Heng;LI Hua-chun(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Yunnan Veterinary and Animal Science Institute,Kunming650224,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2019年第1期1-7,共7页
Chinese Journal of Veterinary Science
基金
国家重点研发计划资助项目(2016YFD0500908)
国家公益性行业(农业)科研专项资助项目(201303035)
