柑橘4种病毒多重PCR检测技术的建立及应用  

Establishment and Application of Multiplex PCR Rapid Detection of Four Citrus Viruses

作　　者：黄爱军[1] 王莹 丁敏 卢占军[1] 易龙[1] HUANG Aijun;WANG Ying;DING Min;LU Zhanjun;YI Long(College of Life Sciences,Gannan Normal University,National Navel Orange Engineering Research Center,Ganzhou,Jiangxi 341000,China)

机构地区：[1]赣南师范大学生命科学学院国家脐橙工程技术研究中心

出　　处：《园艺学报》2019年第8期1616-1622,共7页Acta Horticulturae Sinica

基　　金：江西省重点研发计划项目(20161ACF60016);国家自然科学基金项目(31860488)

摘　　要：柑橘衰退病毒(CTV)、柑橘碎叶病毒(CTLV)、柑橘黄化脉明病毒(CYVCV)和柑橘叶斑驳病毒(CLBV)是影响柑橘生产的重要病毒性病害,建立快速、准确的检测方法是防控该病害的基础。本研究通过筛选针对4种病毒基因组保守区域设计的特异性引物,优化影响多重RT-PCR扩增的引物浓度、退火温度,建立了可同时扩增4种病毒的多重RT-PCR检测体系。该检测体系扩增的特异片段大小分别是CTLV 889 bp、CYVCV 612 bp、CTV 462 bp和CLBV 294 bp,扩增产物清晰,特异,检测灵敏度较普通单重RT-PCR灵敏度低约10倍。采用多重RT-PCR和单重RT-PCR对收集到的59份田间样品分别进行检测,结果显示2种方法检测结果一致,4种病毒的检出率在11.9%~54.2%之间。建立的多重RT-PCR方法可准确、快速、灵敏地检测单一或复合侵染的4种柑橘病毒。Citrus tristeza virus(CTV),Citrus tatter leaf virus(CVLT),Citrus yellow vein clearing viru(s CYVCV),and Citrus leaf blotch viru(s CLBV)are important graft-transmissible pathogens of citrus.Rapid and accurate detection methods are of great significance for the prevention and control of viral diseases.Four compatible sets of primers specific for each virus were designed based on conserved sequences of coat protein gene for multiplex PCR assay.The crucial factors of multiple PCR including primer concentration and annealing temperature were optimized for the highest sensitivity and specificity.Four specific fragments were simultaneously amplified in one PCR reaction.Their molecular weights were determined to be 889(CTLV),612(CYVCV),462(CTV)and 294 bp(CLBV).The sensibility assay showed that the sensibility of this one-step multiplex PCR is about 10 times lower than that of regular single RT-PCR.Finally,this detection system was used to detect these viruses from 59 field samples.The results revealed that the detection rate of these viruses was between 11.9%and 54.2%.In conclusion,this one-step multiplex PCR system is suitable for rapid detection of the 4 citrus viruses from field sampleswith accuracy,rapidity and sensitivity.

关 键 词：柑橘 柑橘衰退病毒 柑橘碎叶病毒 柑橘黄化脉明病毒 柑橘叶斑驳病毒 多重PCR 

分 类 号：S666[农业科学—果树学]