Exploitation of Plant Quarantine System Using Reporter Phage

在pBluescrip上Tn3和Tn5的反向重复序列之间，克隆了编码荧光素酶亚基的鱼类弧菌LuxA-LuxB。该克隆在转座酶(S)存在的情况下，在体内和体外均被证实能转座到目的载体中。以此为供体，在Aquacosmos，Hamamatsu Photonics Co.的荧光图像分析仪上获得了普通大肠杆菌菌株和O157菌株的报告噬菌体。用滨松光电公司专门为本研究设计的轻便灵敏的光子计数器，在3小时内即可检测到特定的目标细菌，灵敏度极高，对单个细菌的检测效率可达90%。样品中土壤、植物材料和活体材料的加入不会降低灵敏度。在这种方法中，我们使用了十四醛作为底物。该化合物的最佳溶剂为水，对荧光素酶的敏感性和稳定性影响最大。用于分离轴突黄单胞菌的报告噬菌体。Citri、青枯雷尔氏菌和淀粉欧文氏菌，将上述人工移动元件重新克隆到广泛寄主范围的质粒中。此外，还检测了大量感染靶细菌后，裂解法检测细胞DNA、蛋白质、碱性磷酸酶和半乳糖苷酶含量的敏感性。该方法可以在较短的时间内以相同的方法检测到特定的细菌，但与报告噬菌体方法相比，其灵敏度约为千分之一。第二种方法的最大优点是不需要分离特定的报告噬菌体。使用宝丽来胶片(ASA20000)也被证明是一种有效的检测方法，但灵敏度变得千分之一。

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.