title: Quality control for quantitative PCR based on amplification compatibility test.
abstract: Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however there is currently no method to allow for reliable control of the amplification process without directly modifying the sample mix. Herein we present a statistical approach based on multivariate analysis of the amplification response data generated in real-time. The amplification trajectory in its most resolved and dynamic phase is fitted with a suitable model. Two parameters of this model, related to amplification efficiency, are then used for calculation of the Z-score statistics. Each studied sample is compared to a predefined reference set of reactions, typically calibration reactions. A probabilistic decision for each individual Z-score is then used to identify the majority of inhibited reactions in our experiments. We compare this approach to univariate methods using only the sample specific amplification efficiency as reporter of the compatibility. We demonstrate improved identification performance using the multivariate approach compared to the univariate approach. Finally we stress that the performance of the amplification compatibility test as a quality control procedure depends on the quality of the reference set.
authors.affiliation: Technical University Munich, Physiology Weihenstephan, Weihenstephaner Berg 3, 85354 Freising-Weihenstephan, Germany. ale@tichopad.de
authors.forename: Ales
authors.initials: A
authors.lastname: Tichopad
authors.forename: Tzachi
authors.initials: T
authors.lastname: Bar
authors.forename: Ladislav
authors.initials: L
authors.lastname: Pecen
authors.forename: Robert R
authors.initials: RR
authors.lastname: Kitchen
authors.forename: Mikael
authors.initials: M
authors.lastname: Kubista
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
mesh_terms: D004247:DNA / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D015999:Multivariate Analysis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012984:Software; D013634:Tannins / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D013634:Tannins; D004247:DNA