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

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Terminal restriction fragment length polymorphism (T-RFLP) is a widely used method for microbial community profiling.
• Potential biases in T-RFLP analysis can affect the accuracy of community structure and diversity assessments.

Purpose of the Study:

• To evaluate the reliability of T-RFLP profiles in reflecting true microbial community structures and diversities.
• To assess the capability of T-RFLP to detect changes in microbial community composition.

Main Methods:

• Utilized defined artificial communities comprising 30 SSU rRNA gene clones from nine bacterial phyla.
• Quantified biases introduced during PCR amplification, enzymatic restriction, and capillary electrophoresis.
• Developed a predictive model for T-RF abundances based on amplification efficiency and fragment size.

Main Results:

• PCR amplification exhibited a maximum variability factor of 3.5 among clones.
• Downstream analyses introduced a maximum bias factor of 4, leading to a total bias factor of 14 in T-RFLP profiles.
• Despite biases affecting absolute structure detection, relative changes in microbial community structures and diversities were reliably captured.

Conclusions:

• T-RFLP analysis is susceptible to significant biases from PCR amplification and downstream processes.
• The method reliably reflects relative shifts in microbial community structure and diversity.
• T-RFLP profiling is a suitable tool for monitoring environmental influences on microbial communities.