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

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 (, 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.