|Budget Amount *help
¥15,000,000 (Direct Cost : ¥15,000,000)
Fiscal Year 2003 : ¥6,300,000 (Direct Cost : ¥6,300,000)
Fiscal Year 2002 : ¥8,700,000 (Direct Cost : ¥8,700,000)
Aim of the present study is to develop a novel molecular method for analyzing microbial community structure in environments, targeting aerobic chemolithotrophic ammonia-oxidizing bacteria(AOB), by quantifying each phylogenetic group comprising a community. AOB play a pivotal role in the global nitrogen cycle, and their monophyletic nature makes them especially suitable for the application of molecular methods. Since their poor growth, community structure of AOB has been analyzed by molecular methods. Recently, PCR has been introduced for quantifying AOB in environmental samples and its potential and reliability has been examined.
In this study, real time PCR method, one of quantitative PCR methods which potentially allow to precisely estimating number of target cells in a sample, was applied to enumerate AOB cells, and was examined if the method can properly estimate AOB. A couple of primer sets which target different genes specific for AOB, and template DNA preparations extracted from several AOB strains and isolates which have relatively diverse phylogenetic positions were employed. Every combination of primer set and strain/isolate provided a standard curve for quantification, and its correlation was very good. However, standard curves obtained from different strains/isolates did not overlap, which would result in incorrect estimations of the exact target numbers when amplifying DNA from mixed communities, depending on the stain selected for creating a standard curve.
We examined the factors affect the shape of standard curve, and elucidated the mechanism of incorrect estimation of the target genes. Based on the results, we specified the combination of types of primer set and template DNA necessary for correct estimation. Based on the results, realistic strategies for expressing abundance of each phylogenetic group of AOB in a community using available techniques were also discussed.