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.
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 (2(30)-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.
Molecular Characterization of Foodborne Pathogens, Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Wyndmoor, PA 19038, USA. peter.irwin@ars.usda.gov
Peter L
PL
Irwin
Ly-Huong T
LH
Nguyen
Chin-Yi
CY
Chen
Gaylen A
GA
Uhlich
George C
GC
Paoli
D001419:Bacteria / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D008433:Mathematics / Q000379:methods*; D008962:Models, Theoretical; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D004259:DNA-Directed DNA Polymerase