Exploitation of Plant Quarantine System Using Reporter Phage

利用报告噬菌体开发植物检疫系统

基本信息

批准号：
11556009
负责人：
TSUYUMU Shinji
金额：
$ 8.58万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

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.

来自颤音渔民的Luxa-LuxB编码荧光素酶亚基的luxa-luxB在pbluescript上的TN3和TN5的倒重复序列之间克隆。该克隆被证实在发生转座酶（S）的情况下将luxa-luxB在体内和体外均转式质粒。使用该供体质粒，借助发光图像分析仪（Aquacosmos，Hamamatsu Photonics Co.）获得了一般大肠杆菌菌株和大肠杆菌菌株和大肠杆菌O157菌株的报告噬菌体。人们证实，使用方便但敏感的光子计数器可以在三个小时内检测到特定的靶细菌，该计数器是由Hamamatsu Photonics Co专门为这项研究设计的。该测试的灵敏度非常出色，并且可以以90％的效率检测到单个细菌。在样品中包含土壤，植物材料和活库并没有降低灵敏度。在这种方法中，我们使用四核作为底物。对这种化合物的最佳溶剂的测试，水是荧光素酶灵敏度和稳定性的最佳Interms。用于隔离Xanthomonas轴突型PV的记者噬菌体。 Citri，Ralstonia solanacearum和Erwinia amylovora，上述人工移动元素被重合为广泛的宿主范围质粒。除这些方法外，还测试了测试对靶细菌大规模感染后使用裂解，检验了检测细胞含量（例如DNA，蛋白质，碱性磷酸酶和数据半乳糖苷酶）的方法的敏感性。通过这种方法，可以在较短的时间内以相同的方式检测到特定的细菌，但是与报告基因噬菌体方法相比，灵敏度大约是千分之一。第二种方法的最大优势是不需要隔离特定的报告基因噬菌体。使用宝丽来膜（ASA20000）的检测也被证明是有效的方法，但灵敏度已成为千分之二的方法。