摘要
为建立南方菜豆花叶病毒(southern bean mosaic virus,SBMV)的快速、简便、高通量检测技术,加强该病毒的口岸检验检疫,以提纯的SBMV粒子为免疫原免疫BALB/C小鼠,利用杂交瘤技术获得3株杂交瘤细胞株19C3、19H9和20G4,其分泌的SBMV腹水单抗效价均达到10-7,且3个单抗与感染SBMV大豆叶片组织粗提液有强烈的特异性免疫反应,而不与感染南方豇豆花叶病毒(southern cowpea mosaic virus,SCPMV)的豇豆、健康的大豆、毛豆、豌豆、蚕豆和菜豆叶片组织粗提液发生免疫反应。以制备的单抗为核心,建立了检测植物中SBMV的ACP-ELISA和dotELISA两种血清学方法。3个单抗中19H9单抗的检测灵敏度最高,以其建立的ACP-ELISA和dot-ELISA方法检测大豆病叶粗提液的灵敏度分别达到1∶163840和1∶10240稀释浓度。利用建立的dot-ELISA方法可从上海口岸截获的大豆种子中检测出SBMV,且该检测结果得到RT-PCR方法验证。表明制备的SBMV单抗及建立的SBMV血清学检测技术可有效用于我国SBMV的口岸检验检疫。
To establish rapid,simple and high-throughput detection techniques for southern bean mosaic virus(SBMV)and to strengthen the port inspection and quarantine of this virus,three hybridoma lines(19C3,19H9 and 20G4)secreting monoclonal antibodies(MAbs)against SBMV were obtained.The titers of ascitic fluids containing MAbs secreted by the three hybridomas were up to 10-7.All the three MAbs reacted strongly and specifically with SBMV in crude extracts from SBMV-infected soybean tissues.A negative reaction was observed when crude extracts were from cowpea plant tissues infected with SBMV,or from a healthy soybean,edamame,pea,vicia faba,or kidney bean plant tissues.Based on the prepared MAbs,two serological detection assays,ACP-ELISA and dot-ELISA were developed for detecting SBMV in plants.Of the three MAbs,19H9 was the most sensitive for detecting SBMV.The detection endpoints of ACP-ELISA and dot-ELISA assays based on MAb 19H9 were up to 1∶163840 and 1∶10240 dilution for SBMV-infected leaf tissue crude extracts,respectively.Using the developed dot-ELISA assay,SBMV infection was detected in soybean seeds intercepted at Shanghai port.The detection result of the dot-ELISA was verified by RT-PCR.This indicated that the prepared MAbs against SBMV and the developed serological detection techniques for SBMV can be effectively used in port inspection and quarantine of SBMV in China.
作者
王亚琴
田沂民
于翠
周雪平
吴建祥
WANG Yaqin;TIAN Yimin;YU Cui;ZHOU Xueping;WU Jianxiang(State Key Laboratory of Rice Biology,Institute of Biotechnology,Zhejiang University,Hangzhou 310058,Zhejiang Province,China;Shanghai Entry-Exit Inspection and Quarantine Bureau,Shanghai 200135,China)
出处
《植物保护学报》
CAS
CSCD
北大核心
2020年第2期347-354,共8页
Journal of Plant Protection
基金
国家重点研发计划(2017YFD0201604,2016YFD0300706)
浙江大学实验技术研究项目(SJS201809)。
