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

本研究的目的是利用每个物种或属特有的16SrRNA序列模式，建立一种海洋细菌，特别是弧菌科各种成员的原位检测和枚举方法。采用生物素DNA探针的非同位素法，采用光致发光法和BCIP/NBT试剂的显色法进行检测。所用细菌包括哈威弧菌和其他4种弧菌、大肠杆菌、嗜水气单胞菌和盐浮异单胞菌。与“通用探针”一起使用的是针对包括哈维弧菌在内的一个群体的两种DNA探针。将菌株置于合适的尼龙膜表面，测试最佳杂交和检测的实验条件。结果表明，一些海洋细菌如盐浮异单胞菌含有催化光致发光试剂分解的物质，产生假阳性光信号。由于膜上的细菌用福尔马林、乙醇和高压灭菌进行了预处理，因此排除了细菌中剩余磷酸酶的可能性。颜色检测法对目标菌株与其他菌株的区分效果较好。然而，对一种特殊的海洋细菌的检测和枚举结果并不令人满意。

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.