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

  1. R.N. Bushon1,
  2. C.M. Kephart1,
  3. G.F. Koltun1,
  4. D.S. Francy1,
  5. F.W. Schaefer III2,
  6. H.D. Alan Lindquist2

Article first published online: 15 DEC 2009

DOI: 10.1111/j.1472-765X.2009.02788.x

Issue

Letters in Applied Microbiology

Letters in Applied Microbiology

Volume 50, Issue 3, pages 276–282, March 2010

Additional Information

How to Cite

Bushon, R.N., Kephart, C.M., Koltun, G.F., Francy, D.S., Schaefer, F.W. and Alan Lindquist, H.D. (2010), Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR. Letters in Applied Microbiology, 50: 276–282. doi: 10.1111/j.1472-765X.2009.02788.x

Author Information

  1. 1

     Ohio Water Microbiology Laboratory, Ohio Water Science Center, U.S. Geological Survey, Columbus, OH, USA

  2. 2

     National Homeland Security Research Center, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, OH, USA

*Rebecca N. Bushon, US Geological Survey, Ohio Water Science Center, Ohio Water Microbiology Laboratory, 6480 Doubletree Avenue, Columbus, OH 43229-1111, USA. E-mail: rnbushon@usgs.gov

Publication History

  1. Issue published online: 8 FEB 2010
  2. Article first published online: 15 DEC 2009
  3. 2009/1109: received 19 June 2009, revised 25 November 2009 and accepted 5 December 2009

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Keywords:

  • analytical/rapid methods;
  • Bacillus;
  • polymerase chain reaction;
  • rapid methods;
  • water

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.

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