Establishment And Application Of Double Fluorescence Labeling T-RFLP Method

Microbial diversity is one of the important indicators in biological evaluation. The application of molecular biology methods can provide a fast and accurate access to microbial diversity. In this study, an improved double fluorescence labeling terminal fragment length polymorphism (T-RFLP) technique was established and was subsequently applied in microbial diversity analysis of eleven sediment samples from Dalinghe River in Liaoning Province, to provide a preliminary biological evaluation in this area.Firstly, the accuracy and sensitivity of double fluorescence labeling T-RFLP method were tested. Both are the same or better than the traditional T-RFLP. By optimizing the analysis parameters, double fluorescence labeling T-RFLP could effectively detect a microbial community with a number more than fifty in a certain sample.Results obtained in the eleven sediment samples from Dalinghe River are as follows:1. Jaccard’s index analysis revealed high similarities in community structure between D4 plot (near outfall) and downstream sampling plots (D5-D9). Moreover, relative higher similarities were found amongst samples with closer geographic position. This phenomena was more obvious in dry season (in May 2011) samples than in wet season (in August 2010) samples.2. Comprehensive analysis on Shannon-Wiener diversity index, Evenness uniformity index, Simpson dominance index and Patrick rich index demonstrated a relatively high level of bacterial richness and stability existed in eleven plots. The values of Shannon-Wiener diversity index were all more than 2, while the values of Evenness uniformity index were all more than 0.9.3. Principal Component Analysis (PCA) revealed dissolved oxygen value (DO) and pH were the major factors influencing bacterial community structure in Dalinghe River sediment samples.4. Dominant bacterial communities analysis of Dalinghe River eleven sediment samples revealed the existence of Bacillus and Clostridium, which are common in solid samples. Additionally, sulfate-reducing related bacteria species, such as Desulfovibrio, Alcaligenes and Acidithiobacillus, were the dominant bacterial communities at outfall plot.