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    林惠娇, 牟桂萍, 滕少娜, 等. 苹果壳色单隔孢溃疡病菌TaqMan实时荧光PCR检测方法[J]. 华南农业大学学报, 2021, 42(2): 65-70. DOI: 10.7671/j.issn.1001-411X.202004031
    引用本文: 林惠娇, 牟桂萍, 滕少娜, 等. 苹果壳色单隔孢溃疡病菌TaqMan实时荧光PCR检测方法[J]. 华南农业大学学报, 2021, 42(2): 65-70. DOI: 10.7671/j.issn.1001-411X.202004031
    shu
    LIN Huijiao, MOU Guiping, TENG Shaona, et al. A real-time fluorescent PCR method for detection of Botryosphaeria stevensii from apple using TaqMan probe[J]. Journal of South China Agricultural University, 2021, 42(2): 65-70. DOI: 10.7671/j.issn.1001-411X.202004031
    Citation: LIN Huijiao, MOU Guiping, TENG Shaona, et al. A real-time fluorescent PCR method for detection of Botryosphaeria stevensii from apple using TaqMan probe[J]. Journal of South China Agricultural University, 2021, 42(2): 65-70. DOI: 10.7671/j.issn.1001-411X.202004031
    shu

    苹果壳色单隔孢溃疡病菌TaqMan实时荧光PCR检测方法

    A real-time fluorescent PCR method for detection of Botryosphaeria stevensii from apple using TaqMan probe

      摘要
    • 摘要:
      目的  针对我国检疫性植物病原真菌苹果壳色单隔孢溃疡病菌Botryosphaeria stevensii，建立实时荧光PCR检测方法。
      方法  根据苹果壳色单隔孢溃疡病菌及其近似种的β微管蛋白基因(β-tubulin基因)保守序列设计1对特异性引物和1条TaqMan MGB探针，分别以病菌DNA和β-tubulin基因靶标序列重组质粒DNA为阳性标准品检验探针的特异性和灵敏度。
      结果  探针BsP267对苹果壳色单隔孢溃疡病菌菌株表现出特异性阳性扩增，且与近缘种及其他常见的果腐病菌无交叉反应，扩增效率为105.858%，探针检测DNA的灵敏度达到1 fg/μL。
      结论  本研究建立的苹果壳色单隔孢溃疡病菌实时荧光PCR检测法具有高特异性和灵敏度，可用于该病害的防控检测和检疫工作。

       

      Abstract:
      Objective  To establish a real-time fluorescent PCR assay for detecting Botryosphaeria stevensii, which is the causal agent of Diplodia canker on apple and is presently subjected to phytosanitary legislation in China.
      Method  A pair of specific primers and a TaqMan MGB probe were designed based on the conserved sequences of β-tubulin genes of B. stevensii and related species. The specificity and sensitivity of the probe were evaluated using DNAs of B. stevensii strains and recombinant plasmid of β-tubulin gene sequence as positive standard, respectively.
      Result  The probe of BsP267 displayed specificity to B. stevensii strains with positive amplification, while there was no crossing reaction with related species and other common fruit rot pathogens. The PCR amplification efficiency was 105.858% and the detection sensitivity of DNA reached 1 fg/μL.
      Conclusion  The real-time PCR method developed in this study can detect B. stevensii from apple with strong specificity and high sensitivity, thus can be used for the prevention, control, detection and quarantine of this disease.

       

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