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Rapid detection system for important plantpathogenic bacteria using luminescent phage

利用发光噬菌体快速检测重要植物病原菌的系统

基本信息

批准号：
15380034
负责人：
TSUYUMU Shinji
金额：
$ 10.24万
依托单位：
Shizuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

For the detection of plant pathogenic bacteria, Ralstonia solanacearum which is a causal agent bacterial blight of Solanaceous plants, we first isolated the phage specifically infect this bacteria. Its genome was sequenced and the subclone containing the putative lysozyme-like encoding gene was obtained. The cassette containing luciferase genes (luxA and luxB) of Vibrio fisherii was inserted into this lysozyme gene. This construct was found to produce light in the presence of its substrate, tetra-decanal. Then, this plasmid was used as the feeder of lux-inserted lysozyme gene by marker exchanged into the phage lysozyme gene. When the constructed reporter phage was infected to various concentration of R. solanacearum, we found that the light production was detected even 100 cells in the sample. At the same time, we tested the applicability of the phage which was labeled with Biotin to use for the detection directly the pathogen after attachment with streptoavidin coupled with peroxidase. We could up to 100 bacterial cells detect by reading chemiluminescent product by the action of peroxidase. This latter method was shown to be equally effective for the detection of this bacteria.For the detection of Xanthomonas axonopodis pv. citri, a causative agent of citrus canker, we integrated the above luxA,B cassette into the gene encoding putative coat protein using similar method. When this reporter phage was used for the detection of this pathogen, we could detect even 100 of this bacteria. We also tried the phage labeled with Ethidium bromide. By infecting with high moi of the labeled phage, we could detect up to 1000 bacteria by measuring the fluorescence of the attached phages on the bacteria.

为了检测植物致病菌——茄枯病菌（Ralstonia solanacearum），我们首先分离出了专门感染该病菌的噬菌体。对其基因组进行了测序，获得了含有推定溶菌酶样编码基因的亚克隆。将含有鱼弧菌荧光素酶基因luxA和luxB的卡带插入该溶菌酶基因中。这种结构被发现在底物四癸醛存在的情况下产生光。然后，将该质粒通过标记交换到噬菌体溶菌酶基因中，作为luxx插入溶菌酶基因的喂料器。将构建的报告噬菌体感染不同浓度的番茄红霉后，我们发现样品中甚至有100个细胞产生光。同时，我们测试了生物素标记噬菌体与链亲和素偶联过氧化物酶附着后直接用于病原体检测的适用性。通过读取过氧化物酶作用下的化学发光产物，可以检测到多达100个细菌细胞。后一种方法被证明对这种细菌的检测同样有效。目的：检测子午黄单胞菌。柑桔溃疡病的病原菌citri，我们用类似的方法将上述luxA，B卡带整合到编码推定外壳蛋白的基因中。当这个报告噬菌体被用来检测这种病原体时，我们甚至可以检测到100个这种细菌。我们还尝试了用溴化乙锭标记的噬菌体。通过对标记噬菌体进行高强度的感染，通过测量附着在细菌上的噬菌体的荧光，我们可以检测到多达1000个细菌。