PMID- 27077046
OWN - NLM
STAT- PubMed-not-MEDLINE
DCOM- 20160414
LR  - 20231111
IS  - 2214-7535 (Print)
IS  - 2214-7535 (Electronic)
IS  - 2214-7535 (Linking)
VI  - 7
DP  - 2016 Mar
TI  - Real-time PCR probe optimization using design of experiments approach.
PG  - 1-8
LID - 10.1016/j.bdq.2015.12.002 [doi]
AB  - 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 mul 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 mul 
      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.
FAU - Wadle, S
AU  - Wadle S
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; 
      Hahn-Schickard Institut fur Mikro-und Informationstechnik, Georges-Koehler-Allee 
      103, 79110 Freiburg, Germany.
FAU - Lehnert, M
AU  - Lehnert M
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Rubenwolf, S
AU  - Rubenwolf S
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
FAU - Zengerle, R
AU  - Zengerle R
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; 
      Hahn-Schickard Institut fur Mikro-und Informationstechnik, Georges-Koehler-Allee 
      103, 79110 Freiburg, Germany; BIOSS-Centre for Biological Signalling Studies, 
      University of Freiburg, 79110 Freiburg, Germany.
FAU - von Stetten, F
AU  - von Stetten F
AD  - Laboratory for MEMS Applications, IMTEK-Department of Microsystems Engineering, 
      University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany; 
      Hahn-Schickard Institut fur Mikro-und Informationstechnik, Georges-Koehler-Allee 
      103, 79110 Freiburg, Germany.
LA  - eng
PT  - Journal Article
DEP - 20151230
PL  - Germany
TA  - Biomol Detect Quantif
JT  - Biomolecular detection and quantification
JID - 101667177
PMC - PMC4827641
OTO - NOTNLM
OT  - Design of experiments
OT  - Mediator probe PCR
OT  - PCR optimization
OT  - Real-time PCR
OT  - Universal reporter
EDAT- 2016/04/15 06:00
MHDA- 2016/04/15 06:01
PMCR- 2015/12/30
CRDT- 2016/04/15 06:00
PHST- 2015/08/26 00:00 [received]
PHST- 2015/12/16 00:00 [revised]
PHST- 2015/12/16 00:00 [accepted]
PHST- 2016/04/15 06:00 [entrez]
PHST- 2016/04/15 06:00 [pubmed]
PHST- 2016/04/15 06:01 [medline]
PHST- 2015/12/30 00:00 [pmc-release]
AID - S2214-7535(15)30013-9 [pii]
AID - 10.1016/j.bdq.2015.12.002 [doi]
PST - epublish
SO  - Biomol Detect Quantif. 2015 Dec 30;7:1-8. doi: 10.1016/j.bdq.2015.12.002. 
      eCollection 2016 Mar.