Introduction:Influenza is an acute respiratory infection caused by Orthomyxoviridae viruses and remains a major global public health concern.Influenza A (INFA) is especially relevant due to its high genetic variability and pandemic potential driven by its mutation ability.Subtyping INFA by qRT-PCR is challenging because targets often share highly similar regions, and single-nucleotide mismatches can reduce probe binding and amplification efficiency.Highly specific TaqMan probes, potentially combined with double-quencher chemistry, may improve signal-to-noise and preserve subtype specificity.In Brazil, INFA is frequently associated with severe acute respiratory infection (SARI), contributing to seasonal peaks and mortality.Objectives: To aim of this study is to evaluate the impact of double-quencher probe approach on sensitivity and signal quality, particularly for targets with high sequence similarity in order to develop a multiplex qRT-PCR assay for INFA subtyping detection.Methodology: Using anonymized data (resolution No. 510/2016) a viral panel from diluted isolated virus was performed to assess analytical performance across virus low-input conditions, including samples with Ct values around 35 for H1N1 and H3N2, tested in duplicate per dilution.Probe sets were designed in two formats: standard single-quencher (SQ) and double-quencher (DQ) for pdmINFA2009, pdmINFA H1N1 and AH3N2 targets.Ct shift (Ct), amplification curve slope/efficiency, baseline fluorescence/background noise, and end-point signal amplitude (Rn/RFU) were predefined as metric assay and comparison.Results: According to in silico analysis, primers and probes set have a balanced conservative and discriminatory positions region for Hemagglutinin A gene with no cross-reactivity under the tested conditions.Multiplex qRT-PCR assay successfully detected Influenza A and its subtypes.Comparative testing of INFApdm09 and AH3 targets with and without DQ probes showed no meaningful differences in baseline-corrected noise and amplification efficiency across the dilution series.In this study, although raw fluorescence analysis indicated that DQ probes reduced baseline noise, this reduction did not translate into a statistically significant overall performance improvement. Conclusion:In conclusion, although the DQ set showed slightly better performance, the difference between results was small and may not justify the additional cost.Therefore, we prioritized the non-DQ set as a more cost-effective option, reserving DQ use only for truly critical targets or those that show a meaningful loss of performance without this modification.
Guimarães et al. (Thu,) studied this question.