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园艺学报 ›› 2018, Vol. 45 ›› Issue (8): 1605-1612.doi: 10.16420/j.issn.0513-353x.2018-0093

• 新技术与新方法 • 上一篇    下一篇

应用DTBIA技术快速检测柑橘黄龙病菌研究

丁 芳1,2,6,王清廉2,徐国亮1,周佳乐1,曾继吾3,严 翔4,夏长秀4,郭 俊5,刘永忠2,洪 霓1,6,王国平1,6,彭抒昂2,*,John S Hartung7,*   

  1. 1华中农业大学植物科学技术学院,湖北省作物病虫害检测与安全控制重点实验室,武汉 430070;2华中农业大学园艺植物生物学教育部重点实验室,武汉 430070;3广东农业科学院果树研究所,广州 510640;4江西省赣州市柑橘研究所,江西赣州 341000;5云南省农业科学院热带亚热带经济作物研究所,云南瑞丽 678600;6华中农业大学农业微生物学国家重点实验室,武汉 430070;7美国农业部分子植物病理学实验室,马里兰 20705
  • 出版日期:2018-08-25 发布日期:2018-08-25
  • 基金资助:
    国家重点研发计划项目(2017YFD0202000);中央高校基本科研业务费专项资金项目(2662016PY099);国家现代农业产业技术体系建设专项资金项目(CARS-26);泉州市燎原计划项目(2016N001)

Research on the Fast Detection of Candidatus Liberibacter Asiaticus with DTBIA

DING Fang1,2,6,WANG Qinglian2,XU Guoliang1,ZHOU Jiale1,ZENG Jiwu3,YAN Xiang4,XIA Changxiu4,GUO Jun5,LIU Yongzhong2,HONG Ni1,6,WANG Guoping1,6,PENG Shu’ang2,*,and John S HARTUNG7,*   

  1. 1Hubei Key Laboratory of Plant Pathology,Huazhong Agricultural University,Wuhan 430070,China;2Key Laboratory of Horticultural Plant Biology(Huazhong Agricultural University),Ministry of Education,Wuhan 430070,China;3Institute of Fruit Trees,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;4Ganzhou Citrus Research Institute,Ganzhou,Jiangxi 341000,China;5Institute of Tropical and Subtropical Cash Crops,Yunnan Academy of Agricultural Sciences,Ruili,Yunnan 678600,China;6State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University,Wuhan 430070,China;7USDA-ARS-MPPL,MD,20705,USA
  • Online:2018-08-25 Published:2018-08-25

摘要: 柑橘黄龙病病原菌为韧皮部限制性寄生菌Candidatus Liberibacter,迄今为止尚未成功获得离体纯培养。因此基于特异性抗体的高通量血清学检测技术迄今尚未开发。本实验室前期研究利用异源表达重组黄龙病菌外膜蛋白OmpCLas免疫动物,制备并获得了特异性多克隆抗体(Anti-OmpCLas-Pab)。本研究利用直接组织印迹免疫法(Direct tissue blot immunoassay,DTBIA)技术对获得的多克隆抗体的检测效果进行了初步评价,证实该抗体可以特异性识别中国不同地理来源的柑橘黄龙病菌分离物;柑橘叶柄、叶片中脉、枝条、根部、果梗和种子中均存在黄龙病菌,其中根部和种子中的病菌含量相对较低,枝条和叶片中脉中的含量较高。该多克隆抗体的获得为后续快速检测试剂盒或试纸条的研发奠定了基础。

关键词: 柑橘, 黄龙病菌, 快速检测, DTBIA, 多克隆抗体, 特异性

Abstract: Citrus Huanglongbing(HLB)is caused by phloem-limited bacteria named Candidatus Liberibacters. Up to date,despite of all kinds of efforts to get pure culture,HLB bacteria still resist in vitro growth. Fast detection especially the high-throughput methods based on the specificity of serological methods has not yet been developed. In our previous work,we constructed fusion OmpCLas protein and heterogonous expressed to get antigen to produce corresponding polyclonal antibody(Pab). In the present study,we carried out experiment on the assessment of the detection efficiency of Anti-OmpCLas-Pab antibody with direct tissue blot immunoassay(DTIBA). Citrus samples collected from different geographical regions were tested. Different organs of petiole,midrib,stem,root,peduncale and seeds were all analyzed. Detection efficiency of both DTBIA and PCR were compared. Our result indicated:Anti- OmpCLas-Pab could specifically recognize HLB bacteria in different isolates originated from different geographical areas in China,including samples from Jiangxi,Fujian,Yunnan,Guangdong and Hainan provinces. Tests with different tissues of petiole,leaf mid rib,stem,root,peduncale and seeds revealed the presence of HLB bacteria. However,less HLB bacteria were found in roots and seeds;higher titer was found in leaf mid-ribs and stems. Our results indicated that Anti-OmpCLas-Pab is a promising antibody for the fast detection of HLB bacteria which laid a basic foundation for the product development on the fast detection kits and dipsticks.

Key words: Citrus, HLB bacteria, fast detection, DTBIA, polyclonal antibody, specificity

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