Exploitation of Plant Quarantine System Using Reporter Phage

在pBluescript上克隆了编码荧光素酶亚基的鱼弧菌luxA-luxB，其序列位于Tn3和Tn5的反向重复序列之间。在体内和体外转座酶存在的情况下，证实该克隆将luxA-luxB转座到目标质粒中。利用该供体质粒，利用荧光图像分析仪（Aquacosmos, Hamamatsu Photonics Co.）获得了一般大肠杆菌菌株和大肠杆菌O157菌株的报告噬菌体。实验证实，使用滨松光电公司专门设计的方便灵敏的光子计数器，可以在3小时内检测到特定的目标细菌。该测试的灵敏度很高，单个细菌的检测效率为90%。样品中含有土壤、植物材料和牲畜均未降低灵敏度。在这种方法中，我们使用四癸醛作为底物。对该化合物的最佳溶剂进行了测试，水是荧光素酶灵敏度和稳定性的最佳条件。分离轴尾黄单胞菌报告噬菌体。将上述人工移动元件克隆到广泛的宿主质粒中。除上述方法外，在大量感染靶菌后，采用裂解法检测DNA、蛋白质、碱性磷酸酶、半乳糖苷酶等细胞内容物的灵敏度。该方法可以在较短的时间内以相同的方式检测到特定的细菌，但与报告噬菌体法相比，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。用宝丽来胶片（ASA20000）检测也是一种有效的方法，但灵敏度仅为千分之一。

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.