title: A quantitative real time PCR method to analyze T cell receptor Vbeta subgroup expansion by staphylococcal superantigens.
abstract: BACKGROUND
  Staphylococcal enterotoxins (SEs), SE-like (SEl) toxins, and toxic shock syndrome toxin-1 (TSST-1), produced by Staphylococcus aureus, belong to the subgroup of microbial superantigens (SAgs). SAgs induce clonal proliferation of T cells bearing specific variable regions of the T cell receptor beta chain (Vbeta). Quantitative real time PCR (qRT-PCR) has become widely accepted for rapid and reproducible mRNA quantification. Although the quantification of Vbeta subgroups using qRT-PCR has been reported, qRT-PCR using both primers annealing to selected Vbeta nucleotide sequences and SYBR Green I reporter has not been applied to assess Vbeta-dependent expansion of T cells by SAgs.
  
  
  METHODS
  Human peripheral blood mononuclear cells were stimulated with various SAgs or a monoclonal antibody specific to human CD3. Highly specific expansion of Vbeta subgroups was assessed by qRT-PCR using SYBR Green I reporter and primers corresponding to selected Vbeta nucleotide sequences.
  
  
  RESULTS
  qRT-PCR specificities were confirmed by sequencing amplified PCR products and melting curve analysis. To assess qRT-PCR efficiencies, standard curves were generated for each primer set. The average slope and R2 of standard curves were -3.3764 +/- 0.0245 and 0.99856 +/- 0.000478, respectively, demonstrating that the qRT-PCR established in this study is highly efficient. With some exceptions, SAg Vbeta specificities observed in this study were similar to those reported in previous studies.
  
  
  CONCLUSIONS
  The qRT-PCR method established in this study produced an accurate and reproducible assessment of Vbeta-dependent expansion of human T cells by staphylococcal SAgs. This method could be a useful tool in the characterization T cell proliferation by newly discovered SAg and in the investigation of biological effects of SAgs linked to pathogenesis.
authors.affiliation: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844, USA. baram717@uidaho.edu
authors.forename: Keun Seok
authors.initials: KS
authors.lastname: Seo
authors.forename: Joo Youn
authors.initials: JY
authors.lastname: Park
authors.forename: David S
authors.initials: DS
authors.lastname: Terman
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Bohach
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics / Q000276:immunology*; D018089:Superantigens / Q000276:immunology*; D016176:T-Lymphocyte Subsets / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D004768:Enterotoxins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D018089:Superantigens