马铃薯S病毒RT-qPCR通用检测体系的建立与应用

doi:10.7668/hbnxb.20191131

摘要/Abstract

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PVS^O和PVS^A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PVS^O和PVS^A重组质粒分别建立标准曲线，检测接种PVS^O和PVS^A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。PVS^O和PVS^A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。PVS^O和PVS^A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PVS^O和PVS^A拷贝数均大于10⁷数量级，均可用于检测，以PVS^O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PVS^O和PVS^A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 PVS^O and PVS^A 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 PVS^O and PVS^A were established with PVS^O and PVS^A standard recombinant plasmid, and the contents of PVS^O and PVS^A in potato leaves, petioles, stems, roots and dormant tubers of potato varieties Youjin and Jizhangshu 12 inoculated with PVS^O and PVS^A 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 PVS^O and PVS^A 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 PVS^O and PVS^A 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 PVS^O and PVS^A in five parts of potato tissues were over 10⁷, and all of them could be detected, with the content of PVS^O 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 PVS^O and PVS^A 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

中图分类号:

S532
S432.4

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.

http://www.hbnxb.net/CN/Y2020/V35/I6/187

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