Quantitative analysis of human and cow-specific 16S rRNA gene markers for assessment of fecal pollution in river waters by real-time PCR

Abstract

The base sequences representing human and cow-specific 16S rRNA gene markers identified in the T-RFLP analysis were recovered from clone libraries. The human and cow-specific primers were designed from these sequences and their specificities were analyzed with fecal DNAs from human, cow, and pig. AllBac primer set showed positive results for all human, cow, and pig samples, while human-specific primer set showed positive result only for human sample but not for cow or pig samples. Likewise, cow-specific primer set showed positive results only for cow sample but not for human or pig samples. Real-time PCR assay with these primers was developed for the identification and quantification of fecal pollution in the river water. The human and cow-specific markers were detected in the order of 9 log(10) copies per gram wet feces which were two orders of magnitude lower than those of total Bacteroidales. For the river water samples, the human-specific marker was detected in 1.7-6.2 log(10) copies/100 ml water which was 2.4-4.9 orders of magnitude lower than those of total Bacteroidales. There was no significant correlation between total Bacteroidales and conventional fecal indicators, but there was a high correlation between Bacteroidales and human-specific marker. This assay could reliably identify and quantify the fecal pollution sources, enabling effective measures in the watersheds and facilitating water quality management.