Comparison of Conventional and Real-Time PCR for Detection of Human and Cattle Host Specific Markers In Surface Water.

Accurate identification of both source and extent of fecal contamination in an impaired watershed is crucial for developing best management practices. Host specific gene sequences of Bacteroidales were used in this study to compare the effectiveness of conventional and real-time PCR for identification of fecal contamination sources in environmental samples. DNA extracted from stream water was amplified with general Bacteroidales primers and human and cattle host specific primers, using conventional PCR and real-time PCR. E. coli was enumerated simultaneously. Both conventional and real-time PCR detected general Bacteroidales 16S rRNA gene markers in all the water samples. E. coli were found in samples as well. The general Bacteroidales gene markers ranged from 366 to 1,289,898 gene copies/100 ml of water, while E. coli numbers varied between 40 to 5340 CFU/100 ml. Human specific Bacteroidales 16S rRNA gene markers were detected in 90% of the water samples by real-time PCR and in 26.6% of the samples by conventional PCR. Only 23% of the samples were above the minimum detection limit of real-time PCR for the cattle specific Bacteroidales markers, while none of the samples were positive in conventional PCR. Furthermore, the human specific Bacteroidales markers were more prevalent during the dry period than the wet period. This study suggests that conventional PCR can be used to detect the presence of general Bacteroidales marker prior to detection and quantification of host specific markers.