Reliability for detecting composition and changes of microbial communities by T-RFLP genetic profiling

Abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis is commonly used for profiling microbial communities in various environments. However, it may suffer from biases during the analytic process. This study addressed the potential of T-RFLP profiles (1) to reflect real community structures and diversities, as well as (2) to reliably detect changing components of microbial community structures. For this purpose, defined artificial communities of 30 SSU rRNA gene clones, derived from nine bacterial phyla, were used. PCR amplification efficiency was one primary bias with a maximum variability factor of 3.5 among clones. PCR downstream analyses such as enzymatic restriction and capillary electrophoresis introduced a maximum bias factor of 4 to terminal restriction fragment (T-RF) signal intensities, resulting in a total maximum bias factor of 14 in the final T-RFLP profiles. In addition, the quotient between amplification efficiency and T-RF size allowed predicting T-RF abundances in the profiles with high accuracy. Although these biases impaired detection of real community structures, the relative changes in structures and diversities were reliably reflected in the T-RFLP profiles. These data support the suitability of T-RFLP profiling for monitoring effects on microbial communities.