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园艺学报 ›› 2017, Vol. 44 ›› Issue (7): 1405-1414.doi: 10.16420/j.issn.0513-353x.2017-0101

• 新技术与新方法 • 上一篇    下一篇

柑橘黄化脉明病毒和衰退病毒的二重RT-PCR检测体系的建立与应用

赵恒燕,关桂静,周常勇,于云奇,王洪苏,李中安,刘金香*   

  1. 西南大学/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:2017-07-25 发布日期:2017-07-25

Establishment and Application of Duplex RT-PCR for the Detection of Citrus yellow vein clearing virus and Citrus tristeza virus

ZHAO Hengyan,GUAN Guijing,ZHOU Changyong,YU Yunqi,WANG Hongsu,LI Zhongan,and LIU Jinxiang*   

  1. Citrus Research Institute,Southwest University/Chinese Academy of Agricultural Sciences,National Citrus Engineering
    Research Center,Chongqing 400712,China
  • Online:2017-07-25 Published:2017-07-25

摘要:

选用柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)和柑橘衰退病毒(Citrus
tristeza virus,CTV)两种病毒外壳蛋白保守序列及内参基因Ubiquitin 的特异性引物,优化影响二重RT-PCR
反应的Mg2+浓度、dNTPs 浓度、引物浓度和退火温度,建立了针对两种病毒的一步法二重RT-PCR 检测
体系。二重RT-PCR 获得CYVCV、CTV 及Ubiquitin 的特异性片段大小分别为614、373 和194 bp,克隆
测序和序列对比结果显示它们与已报道的病毒序列具有较高的同源性。该体系最低能从40 ng · μL-1 总核
酸中检测出CYVCV,从4 ng · μL-1 总核酸中检测出CTV,其灵敏度与单一RT-PCR 检测灵敏度一致。利
用该体系对33 份田间样品进行检测,CYVCV 和CTV 感染率分别为54.5%和66.7%,复合侵染率高达
36.4%。该一步法二重RT-PCR 技术体系可用于大量田间样品中CYVCV 和CTV 的快速同步检测。

关键词: 柑橘, 柑橘黄化脉明病毒, 柑橘衰退病毒, 二重RT-PCR, 检测

Abstract:

Citrus yellow vein clearing virus(CYVCV)and Citrus tristeza virus(CTV),which are
transmittedby insects,are often mixed infectionon citrus plants. Specific primers for RT-PCR were used
according to the nucleotide sequences of coat protein genes(for both CYVCV and CTV)and Ubiquitin
gene to conduct One-step duplex RT-PCR. Simultaneously,key reaction factors such as the concentration
(Mg2+,dNTPs and primers)and annealing temperature were optimized.The specific fragments of 614 bp
(CYVCV),373 bp(CTV)and 194 bp(Ubiquitin)were amplified successfully from the sample by the
duplex RT-PCR system. Cloning and sequencing results showed that they shared high nucleotide identity
with sequences deposited in GenBank. In addition,the sensibility assay showed that this One-step duplex
RT-PCR could detect CYVCV and CTV from 40 ng · μL-1 and 4 ng · μL-1 of total nucleic acids,and its sensitivity was consistent with that of the regular One-step RT-PCR. Finally,this detection system was
used to detect the infection of both CYVCV and CTV from 33 field samples. Results also revealed that the
infection rate of CYVCV and CTV were 54.5% and 66.7%,respectively,and the co-infected rate came out
to be 36.4%. In conclusion,this One-step duplex RT-PCR system was suitable for rapid detection of both
CYVCV and CTV from a large number of field samples.

Key words: citrus, Citrus yellow vein clearing virus, Citrus tristeza virus, duplex RT-PCR, detection