Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples

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Sample quality is a fundamental parameter for the successful diagnosis of respiratory viruses. This parameter depends upon the concentration of epithelial cells. Respiratory samples are usually heterogeneous, which makes relative quantification of the viral load, against the quantity of cells, the most suitable measurement. The quantification of viral load in the field of respiratory viruses is a vital piece of information. Quantification is required from RNA or DNA viral genomes extracted.To design (RT-)PCR assays for reference genes, which show stable expression during viral infection, to be used as cellular controls and cellular quantification tools.Assays were designed for two reference genes: hypoxanthine phosphoribosyltransferase 1 (HPRT1) and ubiquitin C (UBC). The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for this study. The transcriptional activity of the three genes was studied during infection with respiratory syncytial virus and adenovirus. The HPRT1 q(RT-)PCR assay was used on clinical samples.All the analysis methods concluded that the three reference genes were stably expressed during viral infection. The HPRT1 q(RT-)PCR assay indicated that the majority of clinical samples (n=301, 69%) had a cellular load of between 100 and 10,000 cells/PCR. The data showed that the concentration decreased as the age of patient increased.A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples.

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