Virtual quantification of influenza A virus load by real-time RT-PCR
| Autor: | Alessandro Zanetti, Fausto Baldanti, Pier Giulio Conaldi, Cristina Daleno, Antonio Piralla, Susanna Esposito, Elena Pariani |
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| Rok vydání: | 2013 |
| Předmět: |
Adult
Male Adolescent Serial dilution Coefficient of variation Biology Real-Time Polymerase Chain Reaction medicine.disease_cause Virus Influenza quantification Real-time RT-PCR Cycle threshold Virtual quantification tool (VQT) Viral Matrix Proteins Matrix (chemical analysis) Young Adult Virology Influenza A virus medicine Humans Bland–Altman plot Child Aged Aged 80 and over Infant Middle Aged Viral Load Standard curve Infectious Diseases Real-time polymerase chain reaction Child Preschool Female Plasmids |
| Zdroj: | Journal of Clinical Virology. 56:65-68 |
| ISSN: | 1386-6532 |
| DOI: | 10.1016/j.jcv.2012.09.011 |
| Popis: | 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 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. |
| Databáze: | OpenAIRE |
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