Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR
Simultaneous detection and identification of the Xanthomonas species complex associated with tomato bacterial spot using species-specific primers and multiplex PCR
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DOI：
10.1111/j.1365-2672.2012.05431.x
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发表时间：
2012-12-01
影响因子：
4
通讯作者：
Quezado-Duval, A. M.
中科院分区：
文献类型：
--
作者：
Araujo, E. R.;Costa, J. R.;Quezado-Duval, A. M.
目的建立番茄细菌性斑点病黄单胞菌的同时检测和鉴定方法。方法与结果验证了已报道的针对4种巴西番茄细菌性黄单胞菌(Xanthomonas euvesicatoria，Xanthomonas euvesicatoria，X.vesicatoria，X.vesicatoria，X.peranans，X.gardneri，X.gardneri)各50个菌株设计的引物的特异性和敏感性。此外，我们还测试了一种多重聚合酶链式反应方法，用于同时进行物种鉴定。还验证了在病叶样本中直接检测病原菌的可能性。这些引物具有很高的特异性，只扩增出目标DNA。常规聚合酶链式反应对纯化DNA的敏感性为50pgfu L-1，用于细菌悬液分析的敏感性为5×102~5×104cfu ml-1。多重聚合酶链式反应适用于这四种细菌的检测，与常规聚合酶链式反应检测纯化的DNA具有相似的敏感性。当使用细菌悬液时，只有在包括生物扩增步骤(Bio-PCR)的情况下，它的灵敏度才与常规PCR相似。这两种方法都能在有症状的番茄叶片中检测到病原菌。结论利用基于聚合酶链式反应和多重聚合酶链式反应的方法，可以在种水平上对引起番茄细斑病的巴西黄单胞菌进行鉴别和鉴定。这项研究的意义和影响该方法可能是鉴定和检测植物材料中这些病原体的一种可行的替代工具，并可用于植物病理学实验室的常规诊断目的。
Aims To establish protocols for the simultaneous detection and identification of Xanthomonas species causing tomato bacterial spot. Methods and Results We verified the specificity and sensitivity of the previously reported sets of primers designed for strains of the four species of Brazilian tomato bacterial spot xanthomonads, consisting of 30 of Xanthomonas euvesicatoria, 30 of X. vesicatoria, 50 of X. perforans and 50 of X. gardneri. Furthermore, we tested a multiplex PCR protocol for the purpose of concurrent species identification. The possibility of direct detection of the pathogens in diseased leaf samples was also verified. The primers were highly specific, amplifying only target DNA. The sensitivity of the primers in conventional PCR was 50 pg mu l-1 for purified DNA and ranged from 5 x 102 to 5 x 104 CFU ml-1 when bacterial suspensions were analysed. The multiplex PCR was suitable for the detection of all four species and showed similar sensitivity to conventional PCR when tested on purified DNA. When using bacterial suspensions, its sensitivity was similar to conventional PCR only when a biological amplification step (Bio-PCR) was included. Both methods were able to detect the pathogens in symptomatic tomato leaves. Conclusions Brazilian Xanthomonas strains causing tomato bacterial spot can be differentiated and identified at species level by a PCR-based method and by a multiplex PCR. Significance and Impact of the Study This protocol may be a feasible alternative tool for the identification and detection of these pathogens in plant material and may be used for routine diagnostic purposes in plant pathology laboratories.