Standardization of a high-performance RT-qPCR for viral load absolute quantification of influenza A

•Universal human influenza A viral load absolute quantification by RT-qPCR.•Stable, reproducible curve pattern, easy to propagate, low cost and without matrix effects.•Assay with wide dynamic range, high sensitivity, and specificity applicable in different stages of influenza A infection. Influenza...

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Bibliographic Details
Published in:	Journal of virological methods Vol. 301; p. 114439
Main Authors:	Pereira, L.A., Lapinscki, B.A., Debur, M.C., Santos, J.S., Petterle, R.R., Nogueira, M.B., Vidal, L.R.R., De Almeida, S.M., Raboni, S.M.
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 01-03-2022
Subjects:	Absolute quantification Influenza A MIQE Viral load Influenza A Standard MIQE Viral load Absolute quantification
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Summary:	•Universal human influenza A viral load absolute quantification by RT-qPCR.•Stable, reproducible curve pattern, easy to propagate, low cost and without matrix effects.•Assay with wide dynamic range, high sensitivity, and specificity applicable in different stages of influenza A infection. Influenza is an acute viral infectious respiratory disease worldwide, presenting in different clinical forms, from influenza-like illness (ILI) to severe acute respiratory infection (SARI). Although real-time quantitative polymerase chain reaction (qPCR) is already an important tool for both diagnosis and treatment monitoring of several viral infections, the correlation between the clinical aspects and the viral load of influenza is still unclear. This lack of clarity is primarily due to the low accuracy and reproducibility of the methodologies developed to quantify the influenza virus. Thus, this study aimed to develop and standardize a universal absolute quantification for influenza A by reverse transcription-quantitative PCR (RT-qPCR), using a plasmid DNA. The assay showed efficiency (Eff%) 98.6, determination coefficient (R2) 0.998, linear range 10^1 to 10^10, limit of detection (LOD) 6.77, limit of quantification (LOQ) 20.52 copies/reaction. No inter and intra assay variability was shown, and neither was the matrix effect observed. Serial measurements of clinical samples collected at a 72h interval showed no change in viral load. By contrast, immunocompetent patients have a significantly lower viral load than immunosuppressed ones. Absolute quantification in clinical samples showed some predictors associated with increased viral load: (H1N1)pdm09 (0.045); women (p = 0.049) and asthmatics (p = 0.035). The high efficiency, precision, and previous performance in clinical samples suggest the assay can be used as an accurate universal viral load quantification of influenza A. Its applicability in predicting severity and response to antivirals needs to be evaluated.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0166-0934 1879-0984
DOI:	10.1016/j.jviromet.2021.114439