In silico development of a multiplex diagnostic method for pneumonias

Abstract

Objective: this study proposes the in silico design and validation of primers for multiplex quantitative PCR, aiming at rapidly detecting resistance genes. Methods: resistance gene sequences were obtained from NCBI and aligned using ClustalW to identify conserved regions. Primers and probes were designed with OligoArchitect™ Online and evaluated using IDT’s OligoAnalyzer to prevent secondary structures. Results: the blaSHV and ermA genes were selected as targets, with GAPDH as an internal control. Primers and probes showed structural variations, with differences in Tm and ΔG. Some sequences, such as ermA-F and ermA-R, had GC values near the lower limit, while blaSHV-R exhibited multiple hairpin formations. The multiplexing analysis revealed cross-interactions, with ΔG values ranging from -0.2 to -7.2 kcal/mol, indicating variable stability. Conclusion: the designed primers and probes exhibited appropriate GC content, Tm, and Gibbs free energy values, suggesting potential for molecular diagnosis. However, experimental tests are required to confirm their effectiveness in multiplex qPCR.