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

• Published in: Letters in applied microbiology Vol. 50; no. 3; pp. 276 - 282
• Main Authors: Bushon, R.N, Kephart, C.M, Koltun, G.F, Francy, D.S, Schaefer, F.W. III, Alan Lindquist, H.D
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-03-2010; Blackwell Publishing Ltd; Blackwell
• Subjects: analytical/rapid methods; Bacillus; Bacillus anthracis; Bacillus anthracis - genetics; Biological and medical sciences; DNA, Bacterial - analysis; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Francisella tularensis; Francisella tularensis - genetics; Fundamental and applied biological sciences. Psychology; Indicators and Reagents - standards; Microbiology; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - standards; rapid methods; Reagent Kits, Diagnostic - standards; Reagent Kits, Diagnostic - statistics & numerical data; Survival Analysis; Vibrio; Vibrio cholerae - genetics; water; Water; Applied microbiology; rapid methods; Variability; Bacillaceae; Real time; Statistical method; Polymerase chain reaction; Bacillales; DNA; Bacillus; Analytical method; Reagents; Bacteria; Extraction; analytical/rapid methods; Quantitative analysis
• ISSN: 0266-8254; 1472-765X
• DOI: 10.1111/j.1472-765X.2009.02788.x

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. 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. 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. 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.