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柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用

袁琳凯 马崇欢 李丁山 陈志炜 江宵烽 丁新伦 张洁 吴祖建

袁琳凯,马崇欢,李丁山,等. 柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用 [J]. 福建农业学报,2024,39(3):339−344 doi: 10.19303/j.issn.1008-0384.2024.03.011
引用本文: 袁琳凯,马崇欢,李丁山,等. 柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用 [J]. 福建农业学报,2024,39(3):339−344 doi: 10.19303/j.issn.1008-0384.2024.03.011
YUAN L K, MA C H, LI D S, et al. A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants [J]. Fujian Journal of Agricultural Sciences,2024,39(3):339−344 doi: 10.19303/j.issn.1008-0384.2024.03.011
Citation: YUAN L K, MA C H, LI D S, et al. A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants [J]. Fujian Journal of Agricultural Sciences,2024,39(3):339−344 doi: 10.19303/j.issn.1008-0384.2024.03.011

柑橘3种病毒类病原多重RT-PCR检测技术的建立及应用

doi: 10.19303/j.issn.1008-0384.2024.03.011
基金项目: 福建省三明市横向项目(KH210264A、KH210295A、KH220062A)
详细信息
    作者简介:

    袁琳凯(2000 —),男,硕士生,主要从事植物病毒学研究,E-mail:2832507544@qq.com

    马崇欢(1997 — ),男,硕士生,主要从事植物病毒学研究,E-mail:1729919677@qq.com

    通讯作者:

    张洁(1983 —),女,博士,讲师,主要从事植物病毒学研究,E-mail:Jiezhang3553@163.com

  • 中图分类号: S432.1

A Multiplex RT-PCR Assay for Detecting Three Pathogens Infecting Citrus Plants

  • 摘要:   目的  建立柑橘黄化脉明病毒(citrus yellow vein clearing virus, CYVCV)、柑橘衰退病毒(citrus tristeza virus, CTV)和啤酒花矮化类病毒(hop stunt viroid, HSVd)的多重RT-PCR检测体系。  方法  设计多重RT-PCR引物,分析其特异性,确定其最佳浓度比、最适退火温度及灵敏度,在此基础上对福建地区的柑橘样品进行检测。  结果  确定了CYVCV-F/R、CTV-F/R和HSVd-F/R等3对引物的最佳浓度比例为1∶1∶2,最适退火温度为52.9 ℃,灵敏度结果显示该体系可检测模板稀释到10−2的阳性样品。应用该体系对采自福建部分地区的157份柑橘样品进行检测,结果发现,CYVCV、CTV和HSVd的检出率分别为47.1%、56.7%和22.9%。  结论  成功建立了柑橘CYVCV、CTV和HSVd病原的多重RT-PCR检测方法,为该类病害的检测提供准确、快速的检测方法。
  • 图  1  单重RT-PCR扩增结果

    M:D2000Plus DNA分子量标准;1:CYVCV-F/R扩增;2:CTV-F/R扩增;3:HSVd-F/R扩增。

    Figure  1.  Amplification of single RT-PCR

    M: D2000Plus DNA marker; 1: amplified by CYVCV-F/R; 2: amplified by CTV-F/R; 3: amplified by HSVd-F/R.

    图  2  不同引物浓度组合的扩增结果

    M:D2000Plus DNA分子量标准,1~8引物浓度组合参见表1。

    Figure  2.  Amplification of varied concentration ratios of combined primers

    M: D2000Plus DNA marker, 1–8: concentration ratio of combined primers as shown on Table 1.

    图  3  不同退火温度扩增结果

    Figure  3.  Amplification of primer by different annealing temperatures

    图  4  单重及多重RT-PCR灵敏度检测结果

    M:D2000Plus DNA分子量标准,1:100,2:10−1,3:10−2,4:10−3,5:10−4,6:10−5,7:10−6,8:阴性对照。

    Figure  4.  Assay sensitivity of single and multiplex RT-PCR

    M: D2000Plus DNA marker; 1: 100; 2: 10−1; 3: 10−2; 4: 10−3; 5: 10−4; 6: 10−5; 7: 10−6; 8: negative control.

    图  5  多重RT-PCR体系的应用

    A: 多重RT-PCR检测样品; M:D2000Plus DNA分子量标准,1~16:16份柑橘样品,17:阴性对照。B: 检出率分析; C: 复合侵染分析。

    Figure  5.  Application of multiplex RT-PCR assay

    A: Detection by multiplex RT-PCR; M: D2000Plus DNA marker; 1–16: 16 citrus samples; 17: negative control; B: detection rate; C: detection on mixed infections.

    表  1  多重RT-PCR引物浓度组合

    Table  1.   Primers for multiplex RT-PCR assay (单位:μmol·L−1

    引物名称
    Primer name
    引物浓度组合
    Combinations of different primer concentration
    1 2 3 4 5 6 7 8
    CYVCV-F/R 0.2 0.2 0.15 0.15 0.2 0.15 0.15 0.15
    CTV-F/R 0.2 0.1 0.15 0.15 0.15 0.15 0.2 0.2
    HSVd-F/R 0.3 0.15 0.3 0.2 0.3 0.15 0.3 0.2
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-10-25
  • 修回日期:  2024-01-05
  • 网络出版日期:  2024-05-08
  • 刊出日期:  2024-03-28

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