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Mg^(2+)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。
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.
作者
赵恒燕
关桂静
周常勇
于云奇
王洪苏
李中安
刘金香
ZHAOHengyan GUANGuijing ZHOUChangyong YUYunqi WANG Hongsu LIZhongan and LIU Jinxiang(Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, National Citrus Engineering Research Center, Chongqing400712, China)
出处
《园艺学报》
CAS
CSCD
北大核心
2017年第7期1405-1414,共10页
Acta Horticulturae Sinica
基金
西南大学基本科研业务费项目(XDJK2014C131)
国家自然科学基金项目(30600419)
国家现代农业产业技术体系建设专项资金项目
重庆市科委社会事业与民生保障科技创新专项(cstc2016shms-ztzx80003)
作者简介
通信作者Author for correspondence（E—mail：Ljinxiang@126．com；Tel：023—68349017）
