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园艺学报 ›› 2015, Vol. 42 ›› Issue (9): 1843-1850.doi: 10.16420/j.issn.0513-353x.2015-0263

• 新方法 • 上一篇    下一篇

柑橘黄化脉明病毒DTBIA检测方法的建立

宾 羽,宋 震*,李中安,周常勇   

  1. 西南大学柑桔研究所,中国农业科学院柑桔研究所,国家柑桔工程技术研究中心,重庆400712
  • 出版日期:2015-09-25 发布日期:2015-09-25
  • 基金资助:
    农业部公益性行业(农业)科研专项(201203076);重庆市两江学者计划项目;中央高校基本科研专项(SWU113097);重庆市基础及前沿研究项目(CSTC2014JCYJ A8033)

Direct Tissue Blot Immunoassay for Detection of Citrus yellow vein clearing virus

BIN Yu,SONG Zhen*,LI Zhong-an,and ZHOU Chang-yong   

  1. Citrus Research Institute,Southwest University,Chinese Academy of Agricultural Sciences,National Citrus Engineering Research Center,Chongqing 400712,China
  • Online:2015-09-25 Published:2015-09-25

摘要: 通过柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)兔源多克隆抗体(一抗)及Ap标记羊抗兔IgG(二抗)最佳稀释度等条件优化建立了CYVCV直接组织点免疫(Direct tissue blot immunoassay,DTBIA)检测方法,并对其特异性、稳定性和检测效果进行了评价。试验结果显示,一抗、二抗稀释比例分别为1︰8 000和1︰2 000时,DTBIA检测CYVCV效果最佳;应用所建立的DTBIA方法对携带9种不同柑橘病害的柑橘样品进行检测时,仅携带CYVCV样品呈阳性反应;柑橘黄脉病样品植物组织印迹后,印迹膜于4 ℃保存,90 d内可进行有效的CYVCV检测;对田间不同柑橘品种利用DTBIA法检测CYVCV,其结果与一步法RT-PCR符合率为97.92%。可见,该CYVCV检测方法快速、简便、稳定、特异及可靠,可推广应用于田间快速检测CYVCV,对于防范柑橘黄化脉明病毒大面积传播流行具有重要的现实意义。

关键词: 柑橘, 柑橘黄化脉明病毒, 直接组织点免疫法

Abstract: Direct tissue blot immunoassay(DTBIA)was developed to detect Citrus yellow vein clearing virus(CYVCV)by exploring the optimal dilution of rabbit-anti-CYVCV polyclonal antibody (first antibody)and Alkaline phosphatase(Ap)conjugated goat-anti-rabbit IgG(secondary antibody). The DTBIA was the most effective when the first antibody and secondary antibody diluted in 8 000 and 2 000 separately. Using the method,only CYVCV infected samples showed positive among detected samples associated with different citrus diseases;CYVCV could be effectively detected in 90 days after the tissue blotting membranes kept at 4 ℃. The consistency between DTBIA and one-step RT-PCR was 97.92% in CYVCV detection of citrus varieties. The results showed that the DTBIA method is rapid,simple,stable,specific and reliable,and can be applied for rapid detection of CYVCV in the field,which providing the important practical significance for control of the transmission of citrus yellow vein clearing virus and spreading of the disease widely.

Key words: Citrus, Citrus yellow vein clearing virus, direct tissue blot immunoassay

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