Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater

Seurinck, Sylvie, Defoirdt, Tom, Verstraete, Willy and Siciliano, Steven D. (2005) Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater. Environmental microbiology, 7 2: 249-249. doi:10.1111/j.1462-2920.2004.00702.x


Author Seurinck, Sylvie
Defoirdt, Tom
Verstraete, Willy
Siciliano, Steven D.
Title Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater
Journal name Environmental microbiology   Check publisher's open access policy
ISSN 1462-2912
1462-2920
Publication date 2005-02
Year available 2004
Sub-type Article (original research)
DOI 10.1111/j.1462-2920.2004.00702.x
Volume 7
Issue 2
Start page 249
End page 249
Total pages 259
Editor 11
Place of publication Oxford
Publisher Blackwell Science
Language eng
Subject 100204 Environmental Biotechnology Diagnostics (incl. Biosensors)
Abstract The human-specific HF183 Bacteriodes 16S rRNA genetic marker can be used to detect human faecal pollution in water environments. However, there is currently no method to quantify the prevalence of this marker in environmental samples. We developed a real-time polymerase chain reaction (PCR) assay using SYBR Green I detection to quantify this marker in faecal and environmental samples. To decrease the amplicon length to a suitable size for real-time PCR detection, a new reverse primer was designed and validated on human and animal faecal samples. The use of the newly developed reverse primer in combination with the human-specific HF183 primer did not decrease the specificity of the real-time PCR assay but a melting curve analysis must always be included. This new assay was more sensitive than conventional PCR and highly reproducible with a coefficient of variation of less than 1% within an assay and 3% between assays. As the Bacteroides species that carries this human-specific marker has never been isolated, a bacteria real-time assay was used to determine the detection efficiency. The estimated detection efficiency in freshwater ranged from 78% to 91% of the true value with an average detection efficiency of 83 ± 4% of the true value. Using a simple filtration method, the limit of quantification was 4.7 ± 0.3 × 105 human-specific Bacteroides markers per litre of freshwater. The aerobic incubation of the human-specific Bacteroides marker in freshwater for up to 24 days at 4 and 12°C, and up to 8 days at 28°C, indicated that the marker persisted up to the end of the incubation period for all incubation temperatures.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
Advanced Water Management Centre Publications
 
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Created: Mon, 16 Feb 2009, 17:29:37 EST by Judy Dingwall on behalf of Advanced Water Management Centre