摘要
车前草花叶病毒(Plantago asiatica mosaic virus,PlAMV)病是侵染百合等作物的危险性病害,亟待建立其监测防控体系,以防止其扩散传播。根据PlAMV的外壳蛋白(Coat protein,CP)基因的保守序列设计特异性检测引物,通过对引物浓度和反应退火温度进行优化,建立了以CP-2 F/R为特异引物,引物浓度为10μmol·L^(-1),退火温度为59.4℃的高效实时荧光定量PCR检测技术。该检测技术特异性强,灵敏度是RT-PCR的100倍,病毒最低检出浓度为13 copies·μL^(-1)。对75份田间百合样品进行检测,结果显示:RT-qPCR方法检测(29份,38.67%)高于RT-PCR检测出的阳性样品(21份,28.00%)。
As a potentially harmful virus,plantago asiatica mosaic virus(PlA MV)has been reported to infect lilies and other crops in China,so it is urgent to establish a monitoring and control system to prevent its spread.In this study,specific primer pair CP-2F/R were designed based on the conserved region of coat protein(CP)gene of PlAMV.By optimizing concentration of primers and annealing temperature,an efficient real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was established with optimal primer concentration of 10μmol·L^(-1)and optimal annealing temperature of 59.4℃.The sensitivity of this assay was 100 times higher than that of conventional RT-PCR,and the limit of detection by RT-qPCR was 13 copies·μL^(-1).The established RT-qPCR was used to detect PlAMV in 75lily samples,with conventional RT-PCR used as a reference method,and the results showed that more positive samples were detected by RT-qPCR (29,38.67%)than by RT-PCR(21,28.00%).
作者
宋蒙
徐雷锋
曹雨薇
杨盼盼
毕蒙蒙
何国仁
唐玉超
王静
明军
SONG Meng;XU Leifeng;CAO Yuwei;YANG Panpan;BI Mengmeng;HE Guoren;TANG Yuchao;WANG Jing;MING Jun(Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China)
出处
《园艺学报》
CAS
CSCD
北大核心
2021年第12期2497-2505,共9页
Acta Horticulturae Sinica
基金
贵州省科技计划项目(黔科合成果[2019]4235)
国家重点研发计划项目(2019YFD1001805,2019YFD1001002,2018YFD1000405)
中央级公益性科研院所基本科研业务费专项(IVF-BRF2020019)
中国农业科学院创新工程项目
农业部园艺作物生物学与种质创制重点实验室项目。
关键词
百合
车前草花叶病毒
实时荧光定量PCR
lily
plantago asiatica mosaic virus
real-time fluorescence quantitative PCR
