Journal of Nuclear Agricultural Sciences ›› 2015, Vol. 29 ›› Issue (8): 1464-1470.DOI: 10.11869/j.issn.100-8551.2015.08.1464

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Establishment and Application of a Multiplex RT-PCR Detection Method for SPCSV, SPVG and SPFMV Infecting Sweetpotato

LI Huawei1, XU Yongqing1, QIU Sixin1, LIU Zhonghua1, QIU Yongxing1, TANG Hao1, YU Hua2   

  1. 1. Institute of Crop Sciences, Fujian Academy of Agricultural Sciences/Key Laboratory of Crop Germplasm Utilization Between Fujian and Taiwan, Fuzhou, Fujian 350003;
    2. Centrual Laboratory, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350003
  • Received:2014-07-02 Revised:2015-04-28 Online:2015-08-03 Published:2015-08-05

侵染甘薯的SPCSV、SPVG、SPFMV多重RT-PCR检测方法的建立及应用

李华伟1, 许泳清1, 邱思鑫1, 刘中华1, 邱永祥1, 汤浩1, 余华2   

  1. 1. 福建省农业科学院作物研究所/农业部闽台农作物种质资源利用重点开放实验室, 福建 福州 350003;
    2. 福建省农业科学院中心实验室, 福建 福州 350003
  • 通讯作者: 邱思鑫,男,研究员,主要从事甘薯病虫害防控机理研究。E-mail:25273531@qq.com;余华,女,研究员,主要从事甘薯病虫害防控技术研究。E-mail:yuhua@faas.cn。
  • 作者简介:李华伟,男,助理研究员,主要从事植物病毒检测研究。E-mail:hwlpoto@126.com
  • 基金资助:

    现代农业产业技术体系(CARS-11-B-10),福建省财政专项—福建省农业科学院科技创新团队项目(CXTD-1-1301),福建省省属公益类科研院所基本科研专项(2014R1101010-6)

Abstract:

The objective of this study is to develop a multiplex RT-PCR protocol to detect sweetpotato viruses Sweetpotato chlorotic stunt virus (SPCSV), Sweetpotato virus G (SPVG) and Sweetpotato feathery mottle virus (SPFMV) simultaneously. The compatible sets of primers specific for each virus were designed in conserved regions of the gene (hsp70) of SPCSV, the coat protein genes (CP) of SPVG and SPFMV for multiplex RT-PCR. A multiplex RT-PCR(mRT-PCR)system detecting SPCSV,SPVG and SPFMV was established through progressively adjusting and optimizing the RT-PCR conditions. The expected fragments of 304 bp for SPCSV, 433 bp for SPVG and 601 bp for SPFMV were successfully amplified by the multiplex RT-PCR system. Their nucleotide sequences showed 94% to 99% identity with the corresponding sequences of other reported isolates of SPCSV, SPVG and SPFMV.As the multiplex RT-PCR protocol can simultaneously detect SPCSV, SPVG and SPFMV with high stability, accuracy and sensitivity, it can be used to diagnose virus disease in sweetpotato.

Key words: sweetpotato viruses, sweetpotato chlorotic stunt virus, sweeptotato virus G, sweet potato feathery mottle virtus, multiplex RT-PCR, detection method

摘要:

为建立可同时检测甘薯褪绿矮化病毒(SPCSV)、甘薯G病毒(SPVG)和甘薯羽状斑驳病毒(SPFMV)多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因(hsp70)及SPVG、SPFMV外壳蛋白基因(CP)基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%~99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。

关键词: 甘薯病毒, 褪绿矮化病毒, G病毒, 羽状斑驳病毒, 多重RT-PCR, 检测方法