HIRATA Tetsuya Tomonoagrica Co., Institute for Biology, Researcher, 生物研究所, 研究員
MITA Satoru Shizuoka University, Institute for Genetic Research and Biotechnology, Associate Professor, 遺伝子実験施設, 助教授 (20273170)
|Budget Amount *help
¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2000: ¥4,600,000 (Direct Cost: ¥4,600,000)
Fiscal Year 1999: ¥8,800,000 (Direct Cost: ¥8,800,000)
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.