In situ Detection and Enumeration of Marine Bacteria Utilizing 16SrRNA Sequences speciffic to Species and Genera

1993 Fiscal Year Final Research Report Summary

• Project/Area Number: 04454097
• Research Category: Grant-in-Aid for General Scientific Research (B)
• Allocation Type: Single-year Grants
• Research Field: Fisheries chemistry
• Research Institution: HIROSHIMA UNIVERSITY
• Principal Investigator: SIMIDU Usio Hiroshima University, Faculty of Applied Biological Science, Professor, 生物生産学部, 教授 (30101083)
• Co-Investigator(Kenkyū-buntansha): YAMAMOTO Tamiji Hiroshima University, Faculty of Applied Biological Science, Assistant Professor, 生物生産学部, 講師 (40240105) ; MATSUDA Osamu Hiroshima University, Faculty of Applied Biological Science, Professor, 生物生産学部, 教授 (60034469)
• Project Period (FY): 1992 – 1993
• Keywords: 16SrRNA / Vibrionaceae / Marine Bacteria / Hybridization / Detection

Research Abstract

The objective of the research was to establish a method of in situ detection and enumeration of marine bacteria, in particular various members of the family Vibrionaceae utilizing 16SrRNA sequence patterns speciffic to each species or genera. A non isotopic method, which use biotinated DNA probe was applied, and detection was made by a photoluminescens method and a color detection method using BCIP/NBT reagent. The bacteria strains used included Vibrio harveyi and 4 other vibrio species, Escherichia coli and Aeromonas hydrophila and Alteromonas haloplanktis. Two DNA probes that are targeted a group including Vibrio harveyi was used along with the "universal probe." The bacterial strains were applied on the surface of suitable nylon membranes, and experimental conditions for optimal hybridization and detection were tested.
The results showed that some marine bacteria as Alteromonas haloplanktis contain substances that catalyze the decomposition of photoluminescent reagent, producing false positive light signals. The possibility of remaining phosphatase in the bacteria was excluded, since the bacteria on the membrane was pretreated with formalin, ethanol and autoclaving. The color detecting method gave better results for the discrimination of the targeted strains from other strains. However, satisfactory results for the detection and enumeration a particular species of marine bacteria was not obtained.