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A Multiplex Real-Time PCR Assay Differentiates Four Xanthomonas Species Associated with Bacterial Spot of Tomato

机译：多重实时荧光定量PCR检测法可区分四种与番茄细菌斑病相关的黄单胞菌

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摘要

Bacterial spot of tomato, a major. problem in many tomato production areas, is caused by Xanthomonas euvesicatoria, X. vesicatoria, X. peiforans, and X. gardneri. In order to detect and identify the bacterial spot pathogens, we evaluated a region of hipB operon as a source for primers and probes for real-time polymerase chain reaction (PCR). A 420-bp fragment of the hrpB2 gene was amplified by PCR from 75 strains representing the four species. The PCR products were sequenced and phylogenetic analysis revealed that hrpB2 is highly conserved within each species, with a single-nucleotide polymorphism (SNP) among the X. vesicatoria strains. X. euvesicatoria and X. perforans varied by two SNP. Four probes and two primer sets were designed to target the four bacterial spot pathogens based on their hrpB2 gene sequences. In order to simultaneously detect the four bacterial spot pathogens, the four probes and two primer sets were optimized for a multiplex real-time TaqMan PCR assay. The optimized multiplex assay was determined to be highly specific to the four bacterial spot pathogens. Because the optimized multiplex assay facilitated the identification of each bacterial spot pathogen from pure cultures and infected plant tissue, it holds great potential as a diagnostic tool.
机译：番茄细菌斑，主要。在许多番茄产区出现的问题，是由黄单胞菌，黑角菜X. peiforans和黑角菜X. gardneri引起的。为了检测和鉴定细菌斑点病原体，我们评估了hipB操纵子区域作为实时聚合酶链反应（PCR）引物和探针的来源。通过PCR从代表四个物种的75个菌株中扩增了hrpB2基因的420bp片段。对PCR产物进行了测序，系统发育分析表明hrpB2在每个物种内高度保守，在X. vesicatoria菌株中具有单核苷酸多态性（SNP）。 euvesicatoria和X. perforans的变异为两个SNP。设计了四个探针和两个引物组，以基于它们的hrpB2基因序列靶向四种细菌斑病原体。为了同时检测四种细菌斑点病原体，针对多重实时TaqMan PCR分析优化了四种探针和两种引物组。确定优化的多重分析法对四种细菌斑病原体具有高度特异性。由于优化的多重测定法有助于从纯培养物和感染的植物组织中鉴定每种细菌斑点病原体，因此它具有作为诊断工具的巨大潜力。

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