Exploitation of Plant Quarantine System Using Reporter Phage

将编码荧光素酶亚基的luxA-luxB基因克隆到pBluescript上的Tn 3和Tn 5反向重复序列之间。证实该克隆在转座酶存在下在体内和体外都将luxA-luxB转座到靶质粒中。使用该供体质粒，借助于发光图像分析仪（Aquacoptosis，Hamamatsu Photonics Co.）获得普通大肠杆菌菌株和大肠杆菌O 157菌株的报告子序列。经确认，使用Hamamatsu Photonics Co.专门为此研究设计的方便但灵敏的光子计数器，可以在3小时内检测到特定的目标细菌。该试验的灵敏度非常高，可以以90%的效率检测到单个细菌。在样品中包括土壤、植物材料和牲畜并没有降低灵敏度。在该方法中，我们使用十四醛作为底物。对该化合物的最佳溶剂进行了测试，从荧光素酶的灵敏度和稳定性来看，水是最佳溶剂。为了分离出香蕉黄单胞菌致病变种（Xanthomonaxonopodispv.）将上述人工移动的元件重新克隆到广寄主范围质粒中。除这些方法外，还使用裂解法测试了大量感染靶细菌后检测细胞内容物如DNA、蛋白质、碱性磷酸酶和Data-半乳糖苷酶的方法的灵敏度。该方法可以在较短的时间内以同样的方式检测到特定的细菌，但灵敏度约为报告噬菌体方法的千分之一。第二种方法的最大优点是不需要分离特异性报告噬菌体。使用偏振片（ASA 20000）的检测也被证明是有效的方法，但灵敏度为千分之一。

The luxA-luxB from Vibrio fisherii which encode luciferase subunits were cloned between the inverted repeats of Tn3 and Tn5 on pBluescript. This clone was confirmed to transpose luxA-luxB into the target plasmid both in vivo and in vitro in the presence of transposase (s). Using this donor plasmids, the reporter phages for general Eschrichia coli strains and E.coli O157 strains were obtained with the aid of luminescence image analyzer (Aquacosmos, Hamamatsu Photonics Co.). It was confirmed that specific target bacteria could be detected within three hours using the handy but sensitive photon counter which was specifically designed for this study by Hamamatsu Photonics Co. The sensitivity of this test was magnificent, and single bacteria could be detected with 90% efficiency. The inclusion of soil, plant materials and live-stocks in the sample did not lower the sensitivity. In this method, we have used tetra-decanal as the substrate. Test of the optimum solvent for this compound, water was best interms of the sensitivity and stability of luciferase. For the isolation of the reporter phage for Xanthomonas axonopodis pv. citri, Ralstonia solanacearum, and Erwinia amylovora, the above artificial mobile elements were recloned into broad host range plasmid. Besides these approaches, sensitivity of the methods to detect the cellular contents such as DNA, protein, alkaline phosphatase, and Data-galactosidase were tested using lysis-without after massive infection to the target bacteria were tested. By this method, the specific bacteria could be detected in the same manner in shorter period, but the sensitivity was about one thousandth comparing to the reporter phage method. The great advantage of the second method is that isolation of specific reporter phage is not required. The detection using Polaroid film (ASA20000) was also shown to be efficient method, but the sensitivity became one in two thousandth.