title: Real-time PCR probe optimization using design of experiments approach.
abstract: Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.
authors.affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Hahn-Schickard Institut für Mikro-und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Wadle
authors.affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Lehnert
authors.affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Rubenwolf
authors.affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Hahn-Schickard Institut für Mikro-und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; BIOSS-Centre for Biological Signalling Studies, University of Freiburg, 79110 Freiburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Zengerle
authors.affiliation: Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; Hahn-Schickard Institut für Mikro-und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
authors.forename: F
authors.initials: F
authors.lastname: von Stetten
keywords: Design of experiments; Mediator probe PCR; PCR optimization; Real-time PCR; Universal reporter