华北农学报 ›› 2020, Vol. 35 ›› Issue (6): 187-194. doi: 10.7668/hbnxb.20191131

所属专题: 薯类作物

• 资源环境·植物保护 • 上一篇    下一篇

马铃薯S病毒RT-qPCR通用检测体系的建立与应用

程胜群1, 吕文河1, 高艳玲1,2, 白艳菊2, 范国权2, 张威2, 张抒2, 邱彩玲2, 申宇2, 董学志2, 白雅梅3   

  1. 1. 东北农业大学 农学院, 黑龙江 哈尔滨 150030;
    2. 黑龙江省农业科学院 马铃薯研究所, 黑龙江 哈尔滨 150086;
    3. 东北农业大学 资源与环境学院, 黑龙江 哈尔滨 150030
  • 收稿日期:2020-07-12 出版日期:2020-12-28
  • 通讯作者: 吕文河(1960-),男,黑龙江东宁人,教授,博士,主要从事马铃薯遗传育种研究;高艳玲(1977-),女,河北滦南人,副研究员,硕士,主要从事马铃薯病毒研究。
  • 作者简介:程胜群(1994-),女,黑龙江鹤岗人,在读硕士,主要从事马铃薯遗传育种研究。
  • 基金资助:
    黑龙江省农业科学院院级科研项目(2018YYYF022);农业部国家马铃薯产业技术体系(CARS-09-P16);黑龙江省马铃薯产业技术协同创新推广体系(2020)

Development and Application of RT-qPCR Assay for Detection of Potato virus S by Universal Detection System

CHENG Shengqun1, Lü Wenhe1, GAO Yanling1,2, BAI Yanju2, FAN Guoquan2, ZHANG Wei2, ZHANG Shu2, QIU Cailing2, SHEN Yu2, DONG Xuezhi2, BAI Yamei3   

  1. 1. College of Agriculture, Northeast Agricultural University, Harbin 150030, China;
    2. Potato Research Institute, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China;
    3. College of Resources and Environment, Northeast Agricultural University, Harbin 150030, China
  • Received:2020-07-12 Published:2020-12-28

摘要: 为实现准确和快速鉴定PVS及其株系,明确PVS在马铃薯叶片、叶柄、茎、根和休眠块茎中含量以筛选适合的检测部位,设计PVS通用简并引物,建立实时荧光定量PCR(RT-qPCR)技术体系,分析熔解曲线鉴定PVSO和PVSA株系。以马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯M病毒(PVM)、马铃薯A病毒(PVA)和马铃薯卷叶病毒(PLRV)阳性样品为对照检测其特异性。应用PVSO和PVSA重组质粒分别建立标准曲线,检测接种PVSO和PVSA马铃薯品种尤金和冀张薯12号的5个部位PVS含量。另外,应用该体系检测来源于11个省(市、自治区)的90个PVS阳性样品验证其实用性。PVSO和PVSA扩增后熔解曲线分别在85.77~86.00℃和87.78~87.91℃出现特异峰,PVS阴性样品及PVX、PVY、PVM、PVA和PLRV阳性样品的熔解曲线均无上述特异峰。PVSO和PVSA的标准曲线重组质粒浓度分别在1.09×105~1.09×109拷贝/μL和1.26×105~1.26×109拷贝/μL时,荧光信号基线的循环数平均值(Cycle threshold,Ct值)与PVS病毒粒子拷贝数对数之间具有良好的线性关系(决定系数R2=0.994 2和0.991 2)。尤金和冀张薯12号5个部位PVSO和PVSA拷贝数均大于107数量级,均可用于检测,以PVSO在叶柄中含量最高。11个省(市、自治区)的90个PVS阳性样品均可检测到。本研究建立的PVS RT-qPCR检测体系快速、准确、特异,并可依据样品熔解曲线鉴定PVSO和PVSA株系。叶片、叶柄、茎、根和休眠块茎等5个部位组织均可用于检测,实用性强,可为生产脱毒种薯提供技术支持。

关键词: 马铃薯S病毒, 株系, RT-qPCR, 检测, 种薯

Abstract: In order to identify PVS and its individual strains accurately and quickly, and understand the content of PVS in potato leaves, petioles, stems, roots and dormant tubers, which is helpful for selecting suitable detection sites, an Reverse transcription-qPCR (RT-qPCR) was developed by designing a pair of universal primer, and PVSO and PVSA strains were identified using melting curve. The specificity of the system was evaluated with Potato virus X (PVX), Potato virus Y (PVY), Potato virus M (PVM), Potato virus A (PVA) and Potato leafroll virus (PLRV) positive samples. The standard curve of PVSO and PVSA were established with PVSO and PVSA standard recombinant plasmid, and the contents of PVSO and PVSA in potato leaves, petioles, stems, roots and dormant tubers of potato varieties Youjin and Jizhangshu 12 inoculated with PVSO and PVSA were detected. Moreover, ninety positive samples collected from the 11 Provinces (municipality or autonomous region) were verified by the RT-qPCR system. The amplification of PVSO and PVSA strains showed specific peak at 85.77-86.00℃ and 87.78-87.91℃, respectively, in melting curve analysis, but no cross-reaction was found for PVX, PVY, PVM, PVA and PLRV samples. When the concentration of PVSO and PVSA recombinant plasmid were from 1.09×105-1.09×109 copies/μL and 1.26×105-1.26×109 copies/μL, respectively, there was a good linear relationship between the standard curve circulation threshold (Ct) and the log value of PVS virus particles, with determination coefficient being 0.994 2 and 0.991 2, respectively. The content of PVSO and PVSA in five parts of potato tissues were over 107, and all of them could be detected, with the content of PVSO being the highest in petioles. Ninety PVS positive samples from 11 Provinces (municipality or autonomous region) were detected effectively. The PVS RT-qPCR detection system established was fast, accurate and specific and PVSO and PVSA strains could be identified by specific peak in melting curve. The five parts of tissue including leaves, petioles, stems, roots and dormant tubers could be used for detection, with the advantage of good practicability. This research provides technical support to produce virus-free seed potatoes.

Key words: Potato virus S, Strains, RT-qPCR, Detection, Seed potato

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引用本文

程胜群, 吕文河, 高艳玲, 白艳菊, 范国权, 张威, 张抒, 邱彩玲, 申宇, 董学志, 白雅梅. 马铃薯S病毒RT-qPCR通用检测体系的建立与应用[J]. 华北农学报, 2020, 35(6): 187-194. doi: 10.7668/hbnxb.20191131.

CHENG Shengqun, Lü Wenhe, GAO Yanling, BAI Yanju, FAN Guoquan, ZHANG Wei, ZHANG Shu, QIU Cailing, SHEN Yu, DONG Xuezhi, BAI Yamei. Development and Application of RT-qPCR Assay for Detection of Potato virus S by Universal Detection System[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(6): 187-194. doi: 10.7668/hbnxb.20191131.

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