谷物和芸苔黄单胞菌毒力pcr诊断靶基因的选择

E. Kyrova, A. Ignatov
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摘要

2001-2008年在俄罗斯分离到对小麦、黑麦、大麦、番茄、向日葵和芸苔有毒性的植物致病性黄病菌。生理试验和多位点序列分型分析证实了它们属于树黄单胞菌属。从大麦植物中获得的具有代表性的菌株3004的基因组序列草图,也对向日葵、甘蓝和栗子有毒力,证明了3型分泌系统T3SS的缺失,并证明了一些其他毒力基因从远亲细菌中转移的证据。结果表明,T4SS基因可作为新发病原菌群体特异性PCR分析的靶点。提出利用virD4、virB3、virB4和virB9基因设计检测系统。对所选基因进行经典PCR初步实验后,设计引物和TaqMan(R)探针特异性扩增VirD4基因的121 bp片段。扩增产物在所有靶树黄单胞菌菌株中均检测到,而在其他黄单胞菌种类中或在这些寄主植物上发生的其他致病菌或附生细菌中均未检测到扩增产物。该方法可快速检测病株和半选择性培养基上分离的菌落感染,比传统的电镀方法具有更高的灵敏度和特异性。
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Selection of target genes for pcr diagnostics of Xanthomonas arboricola virulent for cereals and brassicas
Plant pathogenic xanthomonads virulent to wheat, rye, barley, tomato, sunflower, and brassicas were isolated in Russia in 2001–2008. Physiological tests and multilocus sequence typing analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of representative strain 3004 from barley plants, which is also virulent to sunflower, brassicas, and chestnut, demonstrated an absence of the Type 3 Secretion System T3SS and an evidence for the lateral gene transfer of some other virulence genes from distantly related bacteria. It was concluded that T4SS genes can be used as the target for group-specific PCR analysis of the emerging pathogen. It was proposed to use virD4, virB3, virB4, and virB9 genes to design a detection system. After preliminary experiments with classic PCR for the chosen genes, primers and TaqMan(R) probe were designed to specifically amplify a 121 bp fragment of the VirD4 gene. Amplification products were obtained for all target Xanthomonas arboricola strains and were not detected in other Xanthomonas species, or in other pathogenic or epiphytic bacteria occurring on these host plants. The assay readily detected Xanthomonas arboricola infection in diseased plants and from bacterial colonies isolated on semi-selective media, and was more sensitive and specific than traditional plating methods.
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