Quantitative polymerase chain reaction (qPCR) is a commonly used technique for plant disease diagnosis, which generally requires DNA extraction from the plant samples. The efficiency of DNA extraction has a significant influence on the accuracy of qPCR. Thus, including a DNA extraction efficiency control is important in qPCR analyses. To develop and evaluate a DNA extraction efficiency control, a (syn. , synonym and correct taxonomic name: ) strain named HUA-22 was created by transferring a green florescent protein (GFP) gene into the genome of the strain Sn15. HUA-22 showed a similar pathogenicity as Sn15 but carried a strong GFP activity. A qPCR primers/probe set named P-GFP, targeting the GFP sequence, was designed. Using P-GFP, qPCR analysis was conducted on DNA extracted from replicated samples of HUA-22 conidia, and confirmed that HUA-22 could be used to characterize the variation in replicated DNA extractions. Conidia of HUA-22 were used to spike soil samples inoculated with the canola clubroot pathogen , canola stem samples infected with the blackleg pathogens and/or and wheat/barley samples infected with pv. () or pv. (). Duplex qPCR using P-GFP and a primers/probe set specific to . , triplex qPCR using P-GFP and primers/probe sets specific to . and , and triplex qPCR using P-GFP and primers/probe sets specific to and were conducted. The results indicated that HUA-22 could be used as a standard for DNA extraction efficiency in qPCR-based plant disease diagnosis. Adding HUA-22 conidia to plant or soil samples prior to DNA extraction, and subsequent use of the P-GFP detection control, provided an added control that could distinguish truly negative from false-negative qPCR results.

中文翻译：

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使用 GFP 标记的 Stagonospora nodorum 菌株作为植物病害诊断中的 DNA 提取效率标准


定量聚合酶链反应（qPCR）是植物病害诊断的常用技术，通常需要从植物样品中提取DNA。 DNA提取效率对qPCR的准确性有显着影响。因此，在 qPCR 分析中纳入 DNA 提取效率控制非常重要。为了开发和评估 DNA 提取效率控制，通过将绿色荧光蛋白 (GFP) 基因转移到菌株 Sn15 的基因组中，创建了名为 HUA-22 的菌株（同义词、同义词和正确的分类名称：）。 HUA-22表现出与Sn15相似的致病性，但具有很强的GFP活性。设计了针对 GFP 序列的名为 P-GFP 的 qPCR 引物/探针组。使用 P-GFP，对从 HUA-22 分生孢子复制样品中提取的 DNA 进行 qPCR 分析，并证实 HUA-22 可用于表征复制 DNA 提取中的变异。将HUA-22的分生孢子掺入接种了双低根肿病病原体的土壤样品、感染黑胫病病原体的双低油菜茎样品和/或感染白斑病菌的小麦/大麦样品。 () 或 pv。 ()。使用 P-GFP 和特定于 的引物/探针组进行双重 qPCR。 ，使用 P-GFP 和特定于 的引物/探针组的三重 qPCR。和 ，并使用 P-GFP 和特异于 和 的引物/探针组进行三重 qPCR。结果表明，HUA-22 可作为基于 qPCR 的植物病害诊断中 DNA 提取效率的标准。在提取 DNA 之前，将 HUA-22 分生孢子添加到植物或土壤样品中，然后使用 P-GFP 检测对照，提供了一个可以区分真阴性和假阴性 qPCR 结果的附加对照。