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Published on: November 1, 2018
Virtual quantification of influenza A virus load by real-time RT-PCR
Antonio Piralla1, Cristina Daleno, Elena Pariani
1Molecular Virology Unit, Virology and Microbiology Department, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy.
Background:
The pan-influenza A real-time RT-PCR detection assay developed by the Centers for Disease Control and Prevention (CDC) during the 2009 pandemic is widely utilized. A quantitative version of the assay may be useful to monitor influenza A infection and response to treatment.
Objectives:
To prove in principle the possibility that a virtual quantification tool (VQT) would allow conversion of CDC real-time RT-PCR cycle threshold (Ct) values in virus RNA copy number.
Study Design:
A plasmid carrying the CDC real-time RT-PCR target region of the influenza A Matrix (M) gene was generated. In a multicenter study, a set of 5 ten-fold dilutions (equivalent to 1×10(2) to 1×10(6)copies/reaction) were prepared and distributed to the 4 participating virology laboratories and then amplified to generate a virtual quantification standard curve. Clinical samples (n=120) were quantified in parallel by interpolation with locally generated standard curves and using the VQT.
Results:
A total of 40 standard curves were obtained by the participating centers (ten from each center). The intra- and inter-laboratory variability showed a coefficient of variation (CV) ≤5%. Influenza A virus quantification in 120 respiratory samples showed a significant correlation between interpolation with locally generated standard curves and the VQT (R(2)=0.9655). Bland Altman analysis showed that the majority (no. 111, 92.5%) of clinical samples had <0.5 log(10) variation.
Conclusions:
VQT proofs the concept that qualitative results from real-time RT-PCR assays can be converted into quantitative determination of virus load in clinical samples without running standard curves in parallel.
