| Budget Amount *help |
¥15,000,000 (Direct Cost: ¥15,000,000)
Fiscal Year 2004: ¥5,700,000 (Direct Cost: ¥5,700,000)
Fiscal Year 2003: ¥9,300,000 (Direct Cost: ¥9,300,000)
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| Research Abstract |
Identification and quantification of microbial contaminants of fecal origin are major priority in the control of drinking and recreational water qualities. In this study, we proposed a new PCR-based approach using 16S rRNA gene markers of enteric anaerobes, Bacteroides-Prevotella spp for discriminating human, cow, and pig fecal contamination in environmental waters without culturing indicator organisms. One human-, three cow-, and two pig-specific Bacteroides-Prevotella 16S rRNA genetic markers were identified based on 16S rRNA gene clone libraries constructed from each fecal samples, respectively. Host-specific markers suggested that there are species composition differences in Bacteroides-Prevotella community in feces of each host species. Additionally, all host-specific genetic markers were detected in river water collected from area frequently contaminated with fecal pollution. We further developed a real-time polymerase chain reaction (qPCR) assay to quantify each host-specific genetic marker in fecal and environmental water samples. First we designed host-specific primers for the real-time PCR assay and validated their specificity on human, cow, and pig fecal samples. The real-time PCR detection of serially diluted DNA extracted DNA from pure cultured B.fragilis and plasmid DNAs was linear ranging from 4.0 to 4.0×10^5 copies/PCR and 6.0×10^1 to 6.0×10^8 copies/PCR, respectively. Using a simple filtration method, the quantification limit of newly developed qPCR assay was 10-70 genetic marker copies/100mL. Second, we applied this qPCR assay with each host-specific primer set to ratural river for over one year and confirmed that our qPCR assay could discriminate human, cow, and pig sources of fecal contamination with high specificity. These results implied that the qPCR assay described here should be widely applicable for monitoring spatial and temporal fluctuations in specific fecal contaminations in natural environments.
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