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首页> 外文期刊>Acta Horticulturae >An immunocapture PCR assay adapted to the detection and the analysis of the molecular variability of apple chlorotic leaf spot virus
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An immunocapture PCR assay adapted to the detection and the analysis of the molecular variability of apple chlorotic leaf spot virus

机译：一种适用于苹果绿萎病叶斑病毒的分子变异性检测和分析的免疫捕获PCR方法

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摘要

A sensitive and polyvalent PCR-based assay was developed for the detection of apple chlorotic leaf spot trichovirus (ACLSV). To achieve max. sensitivity, an immunocapture (IC) step was carried out directly in the PCR tube before the reverse transcription and PCR reactions. In its optimized form, the assay allowed the detection of 10 fg (1 fg=10-12 g) of purified virus, which is equivalent to c. 100 virus particles. The assay was useful for detection of ACLSV in a variety of plant material (leaves,bark, flowers and others) and in a variety of woody hosts including apple, peach, cherry, apricot etc. The results of a full year indexing trial with ELISA and IC-PCR demonstrated an increased rate of ACLSV detection with IC-PCR, especially during the summer period. The IC-PCR was also used, in conjunction with sequencing of the amplified fragment, to study the molecular variability of ACLSV. Extensive sequence divergence between ACLSV isolates was observed. Homology levels of randomly selected isolateswere usually in the range of 80-90%. Most coding differences were observed in the putative viral movement protein, while the coat protein showed better conservation, in keeping with the already known limited serological variability of ACLSV.
机译：开发了一种基于敏感和多价PCR的检测方法，用于检测苹果褪绿叶斑滴虫病毒（ACLSV）。达到最大敏感性方面，在逆转录和PCR反应之前，直接在PCR管中进行了免疫捕获（IC）步骤。以其优化形式，该测定法可检测到10 fg（1 fg = 10-12 g）的纯化病毒，相当于c。 100个病毒颗粒。该测定法可用于检测多种植物材料（叶片，树皮，花朵等）和各种木质宿主（包括苹果，桃，樱桃，杏等）中的ACLSV。ELISA全年索引试验的结果IC-PCR证明用IC-PCR检测ACLSV的比率增加，尤其是在夏季。 IC-PCR还与扩增片段的测序一起用于研究ACLSV的分子变异性。观察到ACLSV分离物之间的广泛序列差异。随机选择的分离株的同源性水平通常在80-90％的范围内。在假定的病毒运动蛋白中观察到大多数编码差异，而外壳蛋白表现出更好的保守性，与已知的ACLSV有限的血清学变异性保持一致。

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