title: Development of an efficient qRT-PCR assay for quality control and cellular quantification of respiratory samples.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  A new tool has been developed and commercialized for quality control and evaluation of cellular concentration in respiratory samples.
authors.affiliation: Normandie University, France; UNICAEN, EA4655 - U2RM, F-14032 Caen, France; bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France. Electronic address: cecile.resa@biomerieux.com.
authors.forename: Cécile
authors.initials: C
authors.lastname: Resa
authors.affiliation: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Magro
authors.affiliation: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Marechal
authors.affiliation: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France.
authors.forename: Côme
authors.initials: C
authors.lastname: Barranger
authors.affiliation: bioMérieux, Parc Technologique Delta Sud, 09340 Verniolle, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Joannes
authors.affiliation: Normandie University, France; UNICAEN, EA4655 - U2RM, F-14032 Caen, France; Department of Virology, University Hospital, F-14033 Caen, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Miszczak
authors.affiliation: Normandie University, France; UNICAEN, EA4655 - U2RM, F-14032 Caen, France; Department of Virology, University Hospital, F-14033 Caen, France.
authors.forename: Astrid
authors.initials: A
authors.lastname: Vabret
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D004267:DNA Viruses / Q000235:genetics; D005786:Gene Expression Regulation; D015967:Gene Expression Regulation, Viral; D020043:Genes, Essential; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D007223:Infant; D008875:Middle Aged; D011786:Quality Control; D012328:RNA Viruses / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics; D019562:Viral Load; D055815:Young Adult
chemical_list: D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D044763:Ubiquitin-Conjugating Enzymes; D007041:Hypoxanthine Phosphoribosyltransferase
keywords: Cellular control; Cellular quantification; Normalization; Respiratory samples; Respiratory viral infections