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园艺学报 ›› 2017, Vol. 44 ›› Issue (5): 999-1004.doi: 10.16420/j.issn.0513-353x.2016-0535

• 新方法 • 上一篇    下一篇

柑橘褪绿矮化相关病毒LAMP检测体系的建立

刘科宏,陈洪明,周 彦,宋 震,李中安*   

  1. 西南大学柑桔研究所,重庆 400712
  • 出版日期:2017-05-25 发布日期:2017-05-25

Establishment of LAMP Assay for Detection of Citrus chlorotic dwarf- associated virus(CCDaV)

LIU Kehong,CHEN Hongming,ZHOU Yan,SONG Zhen,and LI Zhongan *   

  1. Citrus Research Institute,Southwest University,Chongqing 400712,China
  • Online:2017-05-25 Published:2017-05-25

摘要:

根据GenBank登录的柑橘褪绿矮化相关病毒(Citrus chlorotic dwarf-associated virus,CCDaV)的移动蛋白(MP)基因序列设计了一组特异性引物,经体系优化,建立了CCDaV的环介导等温核酸扩增(LAMP)检测方法。结果显示该方法能特异扩增CCDaV,与其他5种柑橘病原物(柑橘衰退病毒、柑橘碎叶病毒、柑橘裂皮病类病毒、温州蜜柑萎缩病毒和柑橘黄龙病菌)不发生反应;灵敏度为PCR的100倍,与实时荧光PCR的灵敏度相同。用LAMP方法对50个田间样品进行检测,与PCR和实时荧光PCR的检测结果一致,证明该方法可用于田间样品的检测。该LAMP检测方法可特异、灵敏、快速地对CCDaV样品进行检测。

关键词: 柑橘, 柑橘褪绿矮化相关病毒, LAMP, 检测

Abstract:

The specific primers of Citrus chlorotic dwarf-associated virus(CCDaV)were designed from the conserved region of movement protein(MP) gene sequence which available in GenBank,and the reaction condition was optimized. A loop-mediated isothermal amplification(RT-LAMP)assay was established for detection of CCDaV. CCDaV was detected while Citrus tristeza virus(CTV),Citrus tatter leaf virus(CTLV),Citrus exocortis viroid(CEVd),Satsuma dwarf virus(SDV)and Huanglongbing(HLB)were not detected by the LAMP assay. Sensitivity of the LAMP assay was 100-fold higher than conventional PCR and same as real-time PCR. The positive rate of 50 samples from Yunnan,Sichuan and Chongqing by using the LAMP was 6%,same as conventional PCR and real-time PCR,suggesting the LAMP assay could detect CCDaV from field samples. The LAMP assay is a specific,sensitive and rapid method for detecting CCDaV.

Key words: Citrus, Citrus chlorotic dwarf-associated virus, LAMP, detection