Qualitative and quantitative estimation of host-specific fecal pollution using Bacteroides–Prevotella 16S rRNA genetic markers by T-RFLP and real-time PCR analyses

Rapid and reliable determination of the non-point sources of fecal pollution is a critical issue for the environmental microbiologists all over the world. In this work we evaluated the use of anaerobic bacterial group Bacteroides–Prevotella as an alternative fecal pollution indicator. Terminal restriction fragment length polymorphism (T-RFLP) and real-time polymerase chain reaction (RT-PCR) analyses were used to monitor and quantify human-, cow- and pig-specific fecal contamination in natural river waters. We also included DNA sequence analysis of the identified fecal markers revealed by T-RFLP in order to clarify the specificity of each marker. It was suggested that the most influent peaks for each fecal source could be used to identify the source of fecal pollution. Development of specific probes based on these markers permit to quantify source of contamination by quantitative RT-PCR. Therefore, we combined the T-RFLP results and RT-PCR assay to quantify fecal contamination by certain host. We can conclude that T-RFLP and RT-PCR analyses showed high reproducibility and sensitivity during analyzing real water samples and can be used to identify, track and quantify host-specific bacterial genetic markers in complex natural water environments.