Real-time PCR probe optimization using design of experiments approach

Biomolecular Detection and Quantification

M. Lehnert¹  S. Rubenwolf¹  F. von Stetten¹  R. Zengerle¹  S. Wadle¹ 

[1] Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;

关键词: Design of experiments;    Mediator probe PCR;    Universal reporter;    Real-time PCR;    PCR optimization;   

DOI  :  10.1016/j.bdq.2015.12.002

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown