扁桃拟茎点霉TaqMan荧光定量PCR检测方法的建立与应用

发布时间：2018-11-24 21:14

【摘要】：扁桃拟茎点霉(Phomopsis amygdali)引起的桃缢缩溃疡病是桃生产上重要的病害之一。带菌苗木及接穗是病害向无病区传播的主要途径。严格的检疫措施是保证无病区健康生产的关键。准确、灵敏、快速的检测方法是严格执行检疫措施及研究病害发生规律的有力工具。将扁桃拟茎点霉组蛋白H3(histone H3)基因与其近缘种比对发现,该基因特异性强,适合作为分子检测的靶标。针对histone H3基因为靶标,设计了特异性引物和Taq Man探针,建立了扁桃拟茎点霉的荧光定量PCR检测方法。结果发现,该方法只能特异识别扁桃拟茎点霉。检测灵敏度可达4×10~1copies·uL~(-1),比常规PCR检测方法高1 000倍;孢子检测最低限达2个孢子,比常规PCR检测方法高10倍,且仅需1 h即可完成检测。该方法用于田间样品检测,扁桃拟茎点霉的阳性检出率达86%,高于分离培养法和常规PCR检出结果。本研究建立的基于Taq Man探针的荧光定量PCR检测体系可用于田间对扁桃拟茎点霉的快速检测。
[Abstract]:Constriction canker caused by (Phomopsis amygdali) is one of the most important diseases in peach production. Bacterial seedlings and scions are the main ways to spread the disease to the disease-free areas. Strict quarantine measures are the key to ensuring healthy production in disease-free areas. Accurate, sensitive and rapid detection method is a powerful tool to strictly carry out quarantine measures and study the regularity of disease occurrence. By comparing the histone H3 (histone H3) gene with its relative species, it was found that the H3 gene was highly specific and suitable for molecular detection. Specific primers and Taq Man probes were designed for the detection of histone H _ 3 as a target, and a fluorescence quantitative PCR method was established for the detection of Pterylocus mandshurica. The results showed that this method could only specifically identify Pteromyces almond. The detection sensitivity is 4 脳 10~1copies uL~ (-1), which is 1 000 times higher than that of the conventional PCR method, and the lowest detection limit of spores is 2 spores, which is 10 times higher than that of the conventional PCR detection method, and the detection can be completed in only 1 hour. The method was applied to the field sample detection. The positive rate of Pteromyces almond was 86%, which was higher than that of culture method and routine PCR. The fluorescent quantitative PCR system based on Taq Man probe can be used for rapid detection of Verticillium almond in the field.
【作者单位】：南京农业大学植物保护学院;农作物生物灾害综合治理教育部重点实验室;无锡出入境检验检疫局;上海农业技术推广服务中心;江苏省植物保护站;江苏省出入境检验检疫局;
【基金】：无锡市科技发展基金(CSE11N1307)
【分类号】：S432.4

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