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园艺学报 ›› 2020, Vol. 47 ›› Issue (7): 1412-1420.doi: 10.16420/j.issn.0513-353x.2019-0859

• 新技术与新方法 • 上一篇    下一篇

莲藕中甘薯潜隐病毒实时荧光定量PCR检测技术的建立及应用

贺 振,董婷婷,吴伟文,陈 雯,李良俊*   

  1. 扬州大学园艺与植物保护学院,江苏扬州 225009
  • 出版日期:2020-07-25 发布日期:2020-07-25
  • 基金资助:
    国家自然科学基金项目(31601604);中央本级重大增减支项目(2060302);国家现代农业产业技术体系建设专项资金项目(CARS-24-B-03)

Development and Evaluation of a Real-time Fluorescent Quantitative PCR Assay for Detection of Sweet Potato Latent Virus-Lotus in Lotus Plants

HE Zhen,DONG Tingting,WU Weiwen,CHEN Wen,and LI Liangjun*   

  1. School of Horticulture and Plant Protection,Yangzhou University,Yangzhou,Jiangsu 225009,China
  • Online:2020-07-25 Published:2020-07-25

摘要: 甘薯潜隐病毒莲藕分离物(sweet potato latent virus-lotus,SPLV-lotus)在江苏省莲藕产区普遍引起发病,并且该分离物序列与已报道的SPLV甘薯分离物存在较大差异。为进一步监测SPLV-lotus在莲藕上的发生情况,针对SPLV-lotus的CP基因设计并优化特异性引物QSPLV-lotus3-F/QSPLV-lotus3-R,通过优化引物浓度和退火温度条件,构建标准曲线,建立了SPLV-lotus实时荧光定量PCR(Real-time fluorescent quantitative polymerase chain reaction,RT-qPCR)检测技术。该方法特异性强,可以快速检测出SPLV-lotus,灵敏度为普通PCR的100倍,可广泛应用于脱毒莲藕SPLV-lotus的精确检测。

关键词: 莲藕, 甘薯潜隐病毒, 实时荧光定量PCR

Abstract: During the survey of lotus viruses disease,sweet potato latent virus-lotus was found to occur at high frequencies in Jiangsu province, China. And there is a significant sequence difference between SPLV-lotus and sweet potato isolates. In order to detect SPLV more accurately,a specific RT-qPCR assay based on SYBR GreenⅠfluorescent dye was established. A specific primer pair QSPLV- lotus3-F/QSPLV-lotus3-R was designed based on the CP gene of SPLV-lotus,and a standard curve was constructed under the optimized primer concentration and annealing temperature. The results showed that the RT-qPCR assay had higher specificity and was 100 times more sensitive for SPLV detection than conventional RT-PCR. This method can be widely used in batch inspection of field samples and be suitable for the detection and identification of virus-free lotus root seedlings.

Key words: lotus, sweet potato latent virus-lotus, RT-qPCR

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