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首页> 外文期刊>Plant Disease >Optimization and Application of a Quantitative Polymerase Chain Reaction Assay to Detect Diaporthe Species in Soybean Plant Tissue
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Optimization and Application of a Quantitative Polymerase Chain Reaction Assay to Detect Diaporthe Species in Soybean Plant Tissue

机译：聚合酶链反应定量检测大豆植物组织中臭虫种类的优化及应用

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摘要

Diaporthe caulivora and D. longicolla are the causal agents of stem canker of soybean (Glycine max L.). Accurate identification of stem canker pathogens upon isolation from infected soybean plants is difficult and unreliable based on morphology. In this study, two TaqMan probe-based quantitative polymerase chain reaction (qPCR) assays were optimized for detection of D. caulivora and D. longicolla in soybean plants. The assays used previously reported D. caulivora-specific (DPC-3) and D. longicolla-specific (PL-3) probe/primer sets. The sensitivity limit of the two assays was determined to be over a range of 100 pg to 10 fg of pure D. caulivora and D. longicolla genomic DNA. The qPCR assays were validated with plant samples collected from commercial soybean fields. The PL-3 set detected D. longicolla in soybean plants collected from the fields (quantification cycle value <35), which was confirmed by isolation on potato dextrose agar (PDA). D. caulivora was detected only in low levels (quantification cycle value <40) by DPC-3 set in a few of the symptomatic field samples, although the pathogen was not isolated on PDA. The qPCR assays were also useful in quantitatively phenotyping soybean plants for resistance to D. caulivora and D. longicolla under greenhouse conditions.
机译：Diaporthe caulivora和D. longicolla是大豆茎溃疡病的病原体（Glycine max L.）。基于形态学，从被感染的大豆植物中分离出来后，准确鉴定茎溃疡病病原是困难且不可靠的。在这项研究中，优化了两个基于TaqMan探针的定量聚合酶链反应（qPCR）分析方法，用于检测大豆植物中的D. caulivora和D. longicolla。先前报道的使用的测定方法是D. caulivora特异（DPC-3）和D. longicolla特异（PL-3）探针/引物组。两种测定的灵敏度极限确定为在100 pg至10 fg的纯D. caulivora和D. longicolla基因组DNA范围内。用从商业大豆田收集的植物样品验证了qPCR分析。 PL-3装置在从田间收集的大豆植物中检测到D. longicolla（定量循环值<35），这通过在马铃薯葡萄糖琼脂（PDA）上分离得到证实。尽管在PDA上未分离到病原体，但通过在一些有症状的野外样品中设置的DPC-3仅检测到了低水平的菜花小球藻（定量循环值<40）。 qPCR分析也可用于定量分析大豆植物在温室条件下对D. caulivora和D. longicolla的抗性。

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