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Published Articles from USDA ARS National Program 108: Food Safety
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Title: Variability in DNA polymerase efficiency: effects of random error, DNA extraction method, and isolate type
Authors: Irwin P, Nguyen LH, Chen CY, He Y
Journal: J of Stat and Math
Accepted date: 2014 Jun 12
Interpretive summary: Foodborne bacteria exist in heterogeneous microbial communities. Some microbes in these communities form multicellular structures and/or biofilms. Pathogens which happen to reside within these structures are more difficult to detect and might be more likely to survive. Part of our research project involves the characterization of such biological constructions from the standpoint of cell number. The only reasonable way for doing this is to count the occurrence of certain genetic components, which are proportional to the number of cells present in a food extract using quantitative, or real time, polymerase chain reaction (qPCR: an analytical method used to determine the number of copies of any particular gene per volume tested). To perform these analyses, DNA standards (various concentrations of known gene copy number) are measured and utilized to convert raw qPCR data associated with unknown test samples into concentration terms. However, the determination of a test sample’s DNA concentration assumes that the concentration dependence (related to reaction efficiency) of the standard and unknown reactions are equivalent. Frequently, this criterion is not met. In this work we have investigated the influence of random error (computer generated), various extraction technologies, and 18 different Gram-positive and -negative foodborne bacteria on PCR efficiency in determining unknown DNA concentration. We found that: even a small amount of random error induces relatively large perturbations in efficiency; most of the variation in efficiency is random albeit some extraction methods/isolates show significant effects on this important qPCR parameter which might be related to polymerase inhibitory compounds being present.
Publication date: 2014 Aug 22
Volume: 1
Issue: 1
Pages: 1-16