| 实时荧光RT-PCR一步法检测苹果茎沟病毒 |
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| 引用本文： | 郭立新,向本春,陈红运,段维军,陈洪俊,朱水芳.实时荧光RT-PCR一步法检测苹果茎沟病毒[J].植物病理学报,2006,36(1):57-61. |
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| 作者姓名： | 郭立新 向本春 陈红运 段维军 陈洪俊 朱水芳 |
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| 作者单位： | 1. 新疆石河子大学农学院植物病理教研室, 石河子 832003;2. 中国检验检疫科学研究院动植物检疫研究所, 北京 100029 |
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| 基金项目： | 教育部全国优秀博士学位论文作者专项基金 |
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| 摘 要： | 根据苹果茎沟病毒(ASGV)各分离物外壳蛋白(CP)基因的保守序列,设计并合成1对特异性引物和1条TaqMan-MGB探针,建屯了对ASGV的实时荧光RT-PCR检测方法。TaqMan-MGB探针3'端具有小沟结合分子(Minor groove binder,MGB),提高了探针的Tm值,缩短了探针长度,解决了病毒各分离物之间基因组变异较大、难以找到较长一致的序列来设计探针的困难。该方法的检测灵敏度比常规RT-PCR电泳检测高约10倍,对于ASGV等在果树体内含量较低的病毒尤为适用。此方法快速、灵敏,整个检测过程完全闭管,无需PCR后处理。
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| 关 键 词： | 苹果茎沟病毒(ASGV) 实时荧光RT-PCR 检测 TAQMAN-MGB探针 |
| 文章编号： | 0412-0914(2006)01-0057-05 |
| 修稿时间： | 2005年2月28日 |
Detection of Apple stem grooving virus by real-time fluorescent RT-PCR one step assay |
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| GUO Li-xin,XIANG Ben-chun,CHEN Hong-yun,DUAN Wei-jun,CHEN Hong-jun,ZHU Shui-fang.Detection of Apple stem grooving virus by real-time fluorescent RT-PCR one step assay[J].Acta Phytopathologica Sinica,2006,36(1):57-61. |
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| Authors: | GUO Li-xin XIANG Ben-chun CHEN Hong-yun DUAN Wei-jun CHEN Hong-jun ZHU Shui-fang |
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| Institution: | 1. Department of Plant Pathology, Institute of Agriculture, Shihezi University, Shihezi 832003, China;2. Institute of Animal and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China |
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| Abstract: | A pair of primers and a TaqMan-MGB probe based on the conserved nucleotide sequence of coat protein (CP) gene of several Apple stem grooving virus (ASGV) isolates were designed and synthesized. A novel real-time fluorescent RT-PCR was established to detect ASGV. The covalent attachment of the minor groove binder moiety at the 3' end of the probe raised the melting temperature to a range suitable for real-time analysis, shortened the probe,and solved the problem of detecting virus with high genome variability among isolates, where an identical sequence can not be found without multiple mismatches. The method was ten times more sensitive than normal RT-PCR and suitable for the detection of those viruses with low concentration in fruit trees. The method was rapid,sensitive and completed within a single tube, without post-PCR handling of the amplification products. |
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| Keywords: | Apple stem grooving virus real-time fluorescent RT-PCR detection TaqMan-MGB probe |
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