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<p>The design of specific primers is a crucial step in developing PCR-based assays for detecting microorganisms, especially when closely related species must be distinguished. In this study, we introduce OligoDesign, a novel software developed to automate and optimize the selection of target-specific primers. The tool aligns input sequences to identify regions of significant divergence, enabling the design of primers with enhanced specificity. Developed in Java, the software integrates sequence alignment, difference detection, and graphical visualization features in a user-friendly desktop application. OligoDesign facilitates the design of up to three oligonucleotides (forward primer, probe, and reverse primer) based on defined regions of sequence divergence, considering essential physicochemical parameters such as melting temperature (T<sub>m</sub>), GC content, and amplicon size. The tool was validated through both in silico analysis and experimental PCR assays using DNA from 22 bacterial strains relevant to food safety. The results confirmed that the designed primers successfully discriminated target from non-target sequences. OligoDesign provides a streamlined, accessible solution for primer design, with potential applications in food diagnostics, clinical microbiology, and molecular ecology.</p>

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OligoDesign: enchancing primer specificity through multi-sequence aligment and validation

  • 期刊论文

  • 发表日期：

    2025年11月12日

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  • Hans Fröder,
  • Guilherme Boschetti,
  • Brenda Picoli Gheno,
  • Pedro Lenz Casa,
  • Tamiris Silva Lopes,
  • Gustavo Sganzerla Martinez,
  • André Luis Martinotto,
  • Scheila de Avila e Silva

摘要

The design of specific primers is a crucial step in developing PCR-based assays for detecting microorganisms, especially when closely related species must be distinguished. In this study, we introduce OligoDesign, a novel software developed to automate and optimize the selection of target-specific primers. The tool aligns input sequences to identify regions of significant divergence, enabling the design of primers with enhanced specificity. Developed in Java, the software integrates sequence alignment, difference detection, and graphical visualization features in a user-friendly desktop application. OligoDesign facilitates the design of up to three oligonucleotides (forward primer, probe, and reverse primer) based on defined regions of sequence divergence, considering essential physicochemical parameters such as melting temperature (Tm), GC content, and amplicon size. The tool was validated through both in silico analysis and experimental PCR assays using DNA from 22 bacterial strains relevant to food safety. The results confirmed that the designed primers successfully discriminated target from non-target sequences. OligoDesign provides a streamlined, accessible solution for primer design, with potential applications in food diagnostics, clinical microbiology, and molecular ecology.