Standardization of a high-performance RT-qPCR for viral load absolute quantification of influenza A pereira2022standardization
Methods/assay standardization study that developed and analytically validated an RT-qPCR absolute quantification assay for influenza A (M gene target) using a plasmid DNA standard. The assay (WHO 2009 M-gene RT-qPCR) was applied to clinical respiratory specimens from a cohort at Hospital de Clínicas, Universidade Federal do Paraná (Curitiba, Brazil). Serial viral-load measurements were obtained for 19 hospitalized SARI patients with two collections (timed by days from symptom onset), including immunocompetent and immunosuppressed participants, to assess viral-load dynamics.
Analytes
respiratory_influenzaA_viral_load
Influenza A viral load by one-step RT-qPCR targeting the influenza A M matrix gene using WHO (2009) primer/probe set on a ViiA 7 Real-Time PCR system. RNA extracted from 200 µL clinical sample (QIAamp Viral RNA Mini Kit). Absolute quantification used a plasmid DNA standard curve (six standards, 10^1 to 10^6 copies/reaction in triplicate). The per-patient serial values here are the absolute quantities tabulated in the paper’s Table 2, in copies/reaction; LOD 6.77 and LOQ 20.52 are reported in the same unit. (The paper additionally converts these to copies/mL and presents Log10 copies/mL in its figures, but the per-patient data used here are the Table 2 copies/reaction values.)
Fitted by censored maximum likelihood. The red line is the median individual; the shaded region is the full range of a simulated cohort drawn from the fitted population, so it shows what simulating from this dataset would produce rather than a confidence interval, with dashed lines at the central 95%. Open triangles are non-detects, drawn at the censoring limit and entering the fit as "below this value" rather than being dropped. See the modelling methods for what these estimates do and do not support.