ThermoPlex: an automated design tool for target-specific multiplex PCR primers based on DNA thermodynamics

Multiplex PCR-based assays are indispensable platforms for rapid and cost-effective DNA-based multi-target detection. The success of such an assay highly depends on the accurate design of oligonucleotide primers, arguably its most vital component. In this study, the ThermoPlex design tool is introduced, offering an automated design pipeline for target-specific multiplex PCR primers motivated by DNA thermodynamics. From a sequence alignment of all relevant target and non-target sequences, ThermoPlex automatically designs multiplex PCR primer candidates in just a matter of minutes. The software also offers tools for thermodynamic calculations that can either be used apart from the automated primer screening routine or in conjunction with other existing primer design tools, depending on the needs of the user. Evidence presented in this study provides insights into the performance of the software performance through theoretical and experimental analyses, serving to establish the reliability of its framework.