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 <0.5log(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.

Virtual quantification of influenza A virus load by real-time RT-PCR / A., Piralla; C., Daleno; E., Pariani; P., Conaldi; Esposito, Susanna Maria Roberta; A. R., Zanetti; F., Baldanti. - In: JOURNAL OF CLINICAL VIROLOGY. - ISSN 1386-6532. - 56:1(2013), pp. 65-68. [10.1016/j.jcv.2012.09.011]

Virtual quantification of influenza A virus load by real-time RT-PCR

Esposito, Susanna Maria Roberta;
2013-01-01

Abstract

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 <0.5log(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.
2013
Virtual quantification of influenza A virus load by real-time RT-PCR / A., Piralla; C., Daleno; E., Pariani; P., Conaldi; Esposito, Susanna Maria Roberta; A. R., Zanetti; F., Baldanti. - In: JOURNAL OF CLINICAL VIROLOGY. - ISSN 1386-6532. - 56:1(2013), pp. 65-68. [10.1016/j.jcv.2012.09.011]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11381/2864694
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