Real-time PCR probe optimization using design of experiments approach
| العنوان: | Real-time PCR probe optimization using design of experiments approach |
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| المؤلفون: | Roland Zengerle, F. von Stetten, Simon Wadle, Michael Lehnert, Stefanie Rubenwolf |
| المصدر: | Biomolecular Detection and Quantification Biomolecular Detection and Quantification, Vol 7, Iss C, Pp 1-8 (2016) |
| بيانات النشر: | Elsevier, 2015. |
| سنة النشر: | 2015 |
| مصطلحات موضوعية: | 0301 basic medicine, Optimal design, lcsh:QR1-502, Biology, PCR optimization, 01 natural sciences, Biochemistry, lcsh:Microbiology, 03 medical and health sciences, Polymerase chain reaction optimization, Structural Biology, Primer dimer, Molecular Biology, lcsh:QH301-705.5, ComputingMethodologies_COMPUTERGRAPHICS, Detection limit, Oligonucleotide, Design of experiments, 010401 analytical chemistry, Molecular biology, 0104 chemical sciences, 030104 developmental biology, Real-time polymerase chain reaction, lcsh:Biology (General), Molecular Medicine, Mediator probe PCR, Primer (molecular biology), Biological system, Universal reporter, Research Paper, Real-time PCR |
| الوصف: | Graphical abstract Highlights • Design of experiments (DOE) is an appropriate approach for probe sequence optimization in real-time PCR. • Nine MP designs led to maximum information for the 3 input factors. • Using DOE the maximum number of individual reactions performed was 180. • A one-factor-at-a-time approach in contrast would require 320 individual reactions. 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. |
| اللغة: | English |
| تدمد: | 2214-7535 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ca554001d0a01039fba170f041f078caTest http://europepmc.org/articles/PMC4827641Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....ca554001d0a01039fba170f041f078ca |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 22147535 |
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