ArticleBiomolecular detection and quantification2016
Real-time PCR probe optimization using design of experiments approach.
Article in Biomolecular detection and quantification, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed, 27 citations in OpenAlex.
- Strategy for Improving the Diagnostic Efficacy of Bovine Tuberculosis Using a Novel PCRVeterinary research communications · 2026Article
- Nanomaterial-nucleic acid probe synergy: accelerating rapid pathogen detection and antimicrobial susceptibility testing in bloodstream infections.Folia microbiologica · 2026Review
- Specificity of Primers and Probes for Molecular Diagnosis ofPathogens (Basel, Switzerland) · 2025Article
- Transfer learning Bayesian optimization for competitor DNA molecule design for use in diagnostic assays.Biotechnology and bioengineering · 2025Article
- Transfer Learning Bayesian Optimization to Design Competitor DNA Molecules for Use in Diagnostic Assays.ArXiv · 2024Article
- Visual Detection of Stem-Loop Primer Amplification (SPA) Products without Denaturation Using Peroxidase-like DNA Machines (PxDM).International journal of molecular sciences · 2023Article
- LNA blockers for improved amplification selectivity.Scientific reports · 2023Article
- Development of In Vitro Dry Eye Models to Study Proliferative and Anti-Inflammatory Effects of Allogeneic Serum Eye Drops.International journal of molecular sciences · 2023Article
- Article
- Development of Hydrolysis Probe-Based qPCR Assays forFoods (Basel, Switzerland) · 2021Article
- Design of Experiments As a Tool for Optimization in Recombinant Protein Biotechnology: From Constructs to Crystals.Molecular biotechnology · 2019Review
- Influence of design probe and sequence mismatches on the efficiency of fluorescent RPA.World journal of microbiology & biotechnology · 2019Article
- Article
- Development of Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Influenza A, B and Adenoviruses.Iranian journal of pathology · 2018Article
- A targeted gene expression platform allows for rapid analysis of chemical-induced antioxidant mRNA expression in zebrafish larvae.PloS one · 2017Article
- Article
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Authors and funding
5 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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