Ivanov V, Tay S T L, Tay J H
School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Ave., Singapore 639798.
Water Sci Technol. 2003;47(5):133-8.
The goal of the research was the development of a simple method to quantify microbial groups in environmental samples. Fluorescence intensity was measured in the sample before and after whole cell fluorescence in situ hybridization with rRNA-targeted, fluorochrome-labeled oligonucleotide probes. To determine specific and non-specific binding of different oligonucleotide probes the following approaches have been used: (1) incubation of the sample with probes at two different temperatures; (2) hybridization of labeled probe in the presence of unlabeled probe; (3) incubation of the sample with labeled specific probe or labeled nonsense probe. Specific binding (hybridization) of the probe was calculated as the difference between total binding and non-specific binding of the probe. Specific binding was 40-50% of total binding in the environmental samples tested. The ratio of the specific binding of different probes may be used to quantify the ratio of different microbial groups in the environmental samples. This quantification is suitable for the microbiological monitoring of microbial aggregates because it is a simple technique and the results can be measured by a portable fluorometer.
该研究的目标是开发一种简单的方法来量化环境样品中的微生物群落。在用靶向rRNA的荧光染料标记寡核苷酸探针进行全细胞荧光原位杂交前后，测量样品中的荧光强度。为了确定不同寡核苷酸探针的特异性和非特异性结合，采用了以下方法：（1）在两个不同温度下将样品与探针孵育；（2）在未标记探针存在的情况下进行标记探针的杂交；（3）将样品与标记的特异性探针或标记的无义探针孵育。探针的特异性结合（杂交）通过探针的总结合与非特异性结合之间的差异来计算。在所测试的环境样品中，特异性结合占总结合的40-50%。不同探针的特异性结合比率可用于量化环境样品中不同微生物群落的比率。这种量化方法适用于微生物聚集体，因为它是一种简单的技术，并且结果可以通过便携式荧光计测量。
