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

利用种属特异性 16SrRNA 序列对海洋细菌进行原位检测和计数

• 批准号: 04454097
• 负责人: SIMIDU Usio
• 金额: $ 3.07万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454097/
• 关键词: 16SrRNA; Vibrionaceae; Marine Bacteria; Hybridization; Detection; 海洋細菌; ビブリオ・ハーベイ; DNAプローブ; ハイブリダイゼーション; 化学発光

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.

本研究的目的是建立一种方法，原位检测和计数的海洋细菌，特别是家庭弧菌科的各种成员利用16 SrRNA序列模式，具体到每个物种或属。采用生物素化DNA探针非同位素法，光致发光法和BCIP/NBT试剂显色法进行检测。所用菌株包括哈维氏弧菌和其他4种弧菌、大肠杆菌、嗜水气单胞菌和盐生交替单胞菌。两种靶向包括哈氏弧菌在内的一组的DNA探针与“通用探针”沿着使用。他说：“我们将细菌菌株涂在合适的尼龙膜表面，并测试最佳杂交和检测的实验条件。结果显示，一些海洋细菌，例如Alteromonas haloorphotis，含有催化光致发光试剂分解的物质，产生假阳性光信号。排除了细菌中残留磷酸酶的可能性，因为膜上的细菌用福尔马林、乙醇和高压灭菌进行了预处理。颜色检测法能较好地区分目标菌株和其它菌株。然而，对于特定种类的海洋细菌的检测和计数没有获得令人满意的结果。