Enhancing the Performance of the CDC Protocol for Real-Time RT-PCR Detection of Influenza A Virus via Post-PCR Melting Curve Analysis

Treewat Watthanachokchai; Kingkan Rakmanee; Pichet Yutthanakarnwikom; Ekawat Pasomsub

doi:10.33165/rmj.48.04.e274725

Authors

Treewat Watthanachokchai Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
Kingkan Rakmanee Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
Pichet Yutthanakarnwikom Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
Ekawat Pasomsub Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand https://orcid.org/0000-0002-4344-0490

DOI:

https://doi.org/10.33165/rmj.48.04.e274725

Keywords:

Real-time RT-PCR, Melting curve analysis, Influenza A virus

Abstract

Background: Influenza has a significant impact on health, society and the economy worldwide.

Objective: To enhance the accuracy and precision of influenza A virus detection.

Methods: Detection of viral genetic material was performed using real-time reverse transcription-polymerase chain reaction (RT-PCR), followed by melting curve analysis of the real-time RT-PCR product.

Results: To enhance diagnostic performance, a novel in-house melting curve analysis assay developed for real-time RT-PCR product, achieving a sensitivity of 96.06% (95% CI, 91.05-98.71) and specificity of 100.00% (95% CI, 95.94-100.00). The assay also demonstrated a positive predictive value of 100.00% (95% CI, 97.02-100.00), a negative predictive value of 94.68% (95% CI, 88.29-97.68) and an accuracy of 97.69% (95% CI, 94.68-99.24). The assay showed high concordance with the NxTAG® Respiratory Pathogen Panel ( $\kappa$ , 0.95; 95% CI, 0.91-0.99; SE, 0.02; P < .001).

Conclusions: This study demonstrated that post-PCR amplicon analysis via melting curve analysis can be performed in a single tube without interference from the hydrolysis probe. Integrating both methods into the same workflow reduces turnaround time for repeat testing, lowers costs, and minimizes labor requirements. As a result, this combined approach improves the efficiency, sensitivity, and specificity of influenza A virus detection, making it a robust tool for laboratory diagnostics.

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