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Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Letters in Applied Microbiology

By:
, , , , ,
DOI: 10.1111/j.1472-765X.2009.02788.x

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Abstract

Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

Additional Publication Details

Publication type:
Article
Publication Subtype:
Journal Article
Title:
Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR
Series title:
Letters in Applied Microbiology
DOI:
10.1111/j.1472-765X.2009.02788.x
Volume
50
Issue:
3
Year Published:
2010
Language:
English
Larger Work Type:
Article
Larger Work Subtype:
Journal Article
First page:
276
Last page:
282
Number of Pages:
7