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.

为了检测植物病原菌青枯雷尔斯顿菌(Ralstonia solanacearum)，它是茄科植物白叶枯病的病原体，我们首先分离出特异性感染该细菌的噬菌体。对其基因组进行了测序，并获得了含有假定的溶菌酶样编码基因的亚克隆。将包含费氏弧菌荧光素酶基因（luxA 和 luxB）的盒插入该溶菌酶基因中。发现该构建体在其底物十四醛存在的情况下产生光。然后，通过将标记交换到噬菌体溶菌酶基因中，将该质粒用作lux插入的溶菌酶基因的饲养层。当构建的报告噬菌体感染不同浓度的青枯菌时，我们发现即使样品中的100个细胞也能检测到光产生。同时，我们测试了生物素标记的噬菌体与链霉亲和素和过氧化物酶结合后用于直接检测病原体的适用性。通过过氧化物酶的作用读取化学发光产物，我们可以检测多达 100 个细菌细胞。后一种方法被证明对于检测这种细菌同样有效。对于检测 Xanthomonas axonopodis pv. citri 是柑橘溃疡病的病原体，我们使用类似的方法将上述 luxA、B 盒整合到编码假定外壳蛋白的基因中。当这个报告噬菌体用于检测这种病原体时，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。通过感染高 moi 的标记噬菌体，我们可以通过测量细菌上附着的噬菌体的荧光来检测多达 1000 种细菌。