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A method for correcting standard-based real-time PCR DNA quantitation when the standard’s polymerase reaction efficiency is significantly different from that of the unknown’s

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Abstract

Standard-based real-time or quantitative polymerase chain reaction quantitation of an unknown sample’s DNA concentration (i.e., [DNA]unk) assumes that the concentration dependence of the standard and unknown reactions (related to reaction efficiency, E) are equivalent. In our work with background food-borne organisms which can interfere with pathogen detection, we have found that it is generally possible to achieve an acceptable E (1 ± 0.05) for standard solutions by optimizing the PCR conditions, template purity, primer sequence, and amplicon lengths. However, this is frequently not true for the solutions containing unknown amounts of target DNA inasmuch as cell extracts are more chemically complex than the standards which have been amplified (230-fold) as well as undergone a purification process. When significant differences in E occur, it is not possible to accurately estimate unknown target DNA concentration from the standard solution’s slope and intercept (from threshold cycle number, or C T , versus Log[DNA] data). What is needed is a standard-mediated intercept which can be specifically coupled with an unknown solution’s PCR concentration dependence. In this work, we develop a simple mathematical procedure to generate a new standard curve with a slope (∂C T /∂Log[Dilution]unk) derived from at least three dilutions of the unknown target DNA solution ([DNA]unk) and an intercept calculated from the unknown’s C T  s, DNA concentrations interpolated from the standard curve (i.e., the traditional estimate of [DNA]unk), and ∂C T /∂Log[Dilution]unk. We were able to achieve this due to our discovery of the predictable way in which the observed and ideal C T versus Log[DNA] slopes and intercepts deviate from one another. This “correction” in the standard-based [DNA]unk determination is typically 20–60% when the difference in the standard and unknown E is >0.1.

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Authors and Affiliations

  1. Molecular Characterization of Foodborne Pathogens, Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA, 19038, USA

    Peter L. Irwin, Ly-Huong T. Nguyen, Chin-Yi Chen, Gaylen A. Uhlich & George C. Paoli

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  1. Peter L. Irwin
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  2. Ly-Huong T. Nguyen
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  3. Chin-Yi Chen
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  4. Gaylen A. Uhlich
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  5. George C. Paoli
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Correspondence to Peter L. Irwin.

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Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned. The USDA is an equal opportunity provider and employer.

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Irwin, P.L., Nguyen, LH.T., Chen, CY. et al. A method for correcting standard-based real-time PCR DNA quantitation when the standard’s polymerase reaction efficiency is significantly different from that of the unknown’s. Anal Bioanal Chem 402, 2713–2725 (2012). https://doi.org/10.1007/s00216-012-5737-9

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  • DOI: https://doi.org/10.1007/s00216-012-5737-9

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