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首页> 外文期刊>Journal of Virological Methods >Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets
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Oligonucleotide-based microarray for detection of plant viruses employing sequence-independent amplification of targets

机译：基于寡核苷酸的微阵列，可利用靶标的序列非依赖性扩增来检测植物病毒

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摘要

The potential of DNA microarrays for detection of plant viruses is hampered by underutilization of sequence-independent amplification methods for target nucleic acid enrichment. A microarray system is described for an unbiased detection of plant viruses using both short (30 nt) and long (50 and 70 nt) oligonucleotide probes. The assay involves amplification of target nucleic acid using random primers followed by in vitro transcription whose cRNA product is labeled chemically, fragmented and used as target for hybridization. Initial optimization tests with Turnip vein clearing virus and Cauliflower mosaic virus showed increased hybridization efficiency with shorter cDNA targets (1100 bp) and longer probes (50 and 70 nt). The system was validated in pure and mixed samples by detection of three Tymovirus species: Asclepias asymptomatic virus, Kennedya yellow mosaic virus and Turnip yellow mosaic virus. The method could detect sequence variants with 70-75% or higher sequence identity, indicating the possible utility of the approach for virus discovery. Array performance comparison of long probes demonstrated the competence of 50-mers to provide a satisfactory balance between detection sensitivity and specificity. The work described is a significant step towards a method to assess, in one assay, the presence of a large diversity of relatives of known viruses of plants.
机译：DNA微阵列检测植物病毒的潜力因未充分利用序列无关的扩增方法来富集目标核酸而受到阻碍。描述了使用短（30 nt）和长（50和70 nt）寡核苷酸探针无偏检测植物病毒的微阵列系统。该测定法包括使用随机引物扩增靶核酸，然后进行体外转录，其cRNA产物经过化学标记，片段化并用作杂交的靶标。芜菁静脉清除病毒和花椰菜花叶病毒的初步优化测试显示杂交效率提高，具有较短的cDNA靶标（1100 bp）和较长的探针（50和70 nt）。该系统已通过检测三种Tymovirus种类（无毒菌，肯尼迪亚黄色花叶病毒和芜菁黄色花叶病毒）在纯样品和混合样品中得到验证。该方法可以检测具有70-75％或更高序列同一性的序列变体，表明该方法可能用于发现病毒。长探针的阵列性能比较证明了50聚体的能力，可以在检测灵敏度和特异性之间提供令人满意的平衡。所描述的工作是朝一种方法评估的重要一步，该方法可以在一种测定中评估植物已知病毒的近亲的多样性。

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