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Evaluation of two spike-and-recovery controls for assessment of extraction efficiency in microbial source tracking studies

Water Research

By:
, ,
DOI: 10.1016/j.watres.2009.06.028

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Abstract

Quantitative PCR (qPCR), applied to complex environmental samples such as water, wastewater, and feces, is susceptible to methodological and sample related biases. In this study, we evaluated two exogenous DNA spike-and-recovery controls as proxies for recovery efficiency of Bacteroidales 16S rDNA gene sequences (AllBac and qHF183) that are used for microbial source tracking (MST) in river water. Two controls-(1) the plant pathogen Pantoea stewartii, carrying the chromosomal target gene cpsD, and (2) Escherichia coli, carrying the plasmid-borne target gene DsRed2-were added to raw water samples immediately prior to concentration and DNA extraction for qPCR. When applied to samples processed in replicate, recovery of each control was positively correlated with the observed concentration of each MST marker. Adjustment of MST marker concentrations according to recovery efficiency reduced variability in replicate analyses when consistent processing and extraction methodologies were applied. Although the effects of this procedure on accuracy could not be tested due to uncertainties in control DNA concentrations, the observed reduction in variability should improve the strength of statistical comparisons. These findings suggest that either of the tested spike-and-recovery controls can be useful to measure efficiency of extraction and recovery in routine laboratory processing. ?? 2009 Elsevier Ltd.

Additional Publication Details

Publication type:
Article
Publication Subtype:
Journal Article
Title:
Evaluation of two spike-and-recovery controls for assessment of extraction efficiency in microbial source tracking studies
Series title:
Water Research
DOI:
10.1016/j.watres.2009.06.028
Volume
43
Issue:
19
Year Published:
2009
Language:
English
Larger Work Type:
Article
Larger Work Subtype:
Journal Article
First page:
4820
Last page:
4827
Number of Pages:
8