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园艺学报 ›› 2015, Vol. 42 ›› Issue (7): 1400-1408.doi: 10.16420/j.issn.0513-353x.2015-0161

• 新方法 • 上一篇    下一篇

苹果茎痘病毒TaqMan探针实时荧光定量RT-PCR检测方法的建立

秦子禹,孙建设,王娜,邵建柱   

  1. 1河北农业大学园艺学院,河北保定 071001;2河北科技师范学院园艺科技学院,河北昌黎 066600
  • 出版日期:2015-07-25 发布日期:2015-07-25
  • 基金资助:

    国家现代农业产业技术体系建设专项资金项目(CARS-28)

Development of a TaqMan Real-time RT-PCR Method for Detecting Apple stem pitting virus(ASPV)

QIN Zi-Yu, SUN Jian-She, WANG Na, SHAO Jian-Zhu   

  1. 1College of Horticulture,Agricultural University of Hebei,Baoding,Hebei 071000,China;2Horticultural College of Science & Technology,Hebei Normal University of Science & Technology,Changli,Hebei 066600,China
  • Online:2015-07-25 Published:2015-07-25

摘要: 为建立一种检测苹果茎痘病毒(Apple stem pitting virus,ASPV)的TaqMan探针实时荧光定量RT-PCR方法,根据ASPV外壳蛋白基因(coat protein,cp)保守序列设计了特异性引物和TaqMan探针,以体外转录的RNA为标准品构建标准曲线,并对该方法的特异性、灵敏性、重复性进行检验。建立的标准曲线决定系数达0.996,扩增效率为99%;该方法特异性好,与苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)、苹果锈果类病毒(ASSVd)均无交叉反应;其最低检测限为1.31 × 102 copies ? μL-1,灵敏度比常规RT-PCR高100倍;批内和批间变异系数均小于1.85%。该方法具有特异性强、灵敏度高、重复性好等优点,适用于田间样品中ASPV的快速定量检测。

关键词: 苹果茎痘病毒, TaqMan 探针, 实时荧光定量RT-PCR

Abstract: This study established a TaqMan real-time RT-PCR method for detecting Apple stem pitting virus(ASPV). A pair of primers and a TaqMan probe were designed based on the conserved nucleotide sequence of ASPV coat protein gene(cp). The RNA standard of ASPV was prepared with transcription in vitro and the standard curve was plotted. The specificity,sensitivity and reproducibility of this method were evaluated. The results showed that the coefficient of determination(R2)was 0.996 and the amplification efficiency(E)was 99%. There was no crossing reaction with Apple stem grooving virus(ASGV),Apple chlorotic leaf spot virus(ACLSV)and Apple scar skin viroid(ASSVd),indicating a strong specificity. The detection limit of this method was 1.31 × 102 copies ? μL-1 and the sensitivity was 100 times higher than the conventional RT-PCR. The coefficients of variation between the intra- and inter-assay were both within 1.85%. This method could be used to detect ASPV rapidly and quantitatively in field samples with strong specificity,high sensitivity and reliable reproducibility.

Key words: Apple stem pitting virus(ASPV), TaqMan probe, real-time RT-PCR

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