Title |
Interlaboratory Comparison of Real-Time PCR Protocols for Quantification of General Fecal Indicator Bacteria
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Published in |
Environmental Science & Technology, December 2011
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DOI | 10.1021/es2031455 |
Pubmed ID | |
Authors |
Orin C. Shanks, Mano Sivaganesan, Lindsay Peed, Catherine A. Kelty, A. Denene Blackwood, Monica R. Greene, Rachel T. Noble, Rebecca N. Bushon, Erin A. Stelzer, Julie Kinzelman, Tamara Anan’eva, Christopher Sinigalliano, David Wanless, John Griffith, Yiping Cao, Steve Weisberg, Valarie J. Harwood, Christopher Staley, Kevin H. Oshima, Manju Varma, Richard A. Haugland |
Abstract |
The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol. |
X Demographics
Geographical breakdown
Country | Count | As % |
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Unknown | 2 | 100% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 1 | 50% |
Scientists | 1 | 50% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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United States | 1 | 1% |
Slovenia | 1 | 1% |
South Africa | 1 | 1% |
Unknown | 69 | 96% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 22 | 31% |
Student > Ph. D. Student | 17 | 24% |
Student > Master | 8 | 11% |
Student > Bachelor | 7 | 10% |
Other | 4 | 6% |
Other | 9 | 13% |
Unknown | 5 | 7% |
Readers by discipline | Count | As % |
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Agricultural and Biological Sciences | 25 | 35% |
Environmental Science | 14 | 19% |
Engineering | 5 | 7% |
Immunology and Microbiology | 5 | 7% |
Biochemistry, Genetics and Molecular Biology | 2 | 3% |
Other | 9 | 13% |
Unknown | 12 | 17% |