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Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets

Environmental Science and Technology

By:
and
DOI: 10.1021/es800051z

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Abstract

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 101 to 108 copies/??l. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 ?? 107 copies/g) to thatfound in human feces (1.1 ?? 107 copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 ?? 104 copies/100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/100 mL water.

Additional Publication Details

Publication type:
Article
Publication Subtype:
Journal Article
Title:
Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets
Series title:
Environmental Science and Technology
DOI:
10.1021/es800051z
Volume
42
Issue:
14
Year Published:
2008
Language:
English
Larger Work Type:
Article
Larger Work Subtype:
Journal Article
Larger Work Title:
Environmental Science and Technology
First page:
5229
Last page:
5234