title: GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus.
abstract: BACKGROUND
  Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions.
  
  
  OBJECTIVE
  We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously.
  
  
  METHODS
  DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured.
  
  
  RESULTS
  Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (kappa=0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (rho(c)=0.95) which validated the proposed strategy.
  
  
  CONCLUSIONS
  We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously.
authors.affiliation: Bauru School of Dentistry, University of São Paulo, Al. Octávio Pinheiro Brisola, 9-75, Bauru, São Paulo 17012-901, Brazil.
authors.forename: Melissa Rodrigues
authors.initials: MR
authors.lastname: de Araujo
authors.forename: Laura
authors.initials: L
authors.lastname: De Marco
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Santos
authors.forename: Izabel Regina Fisher
authors.initials: IR
authors.lastname: Rubira-Bullen
authors.forename: Guglielmo
authors.initials: G
authors.lastname: Ronco
authors.forename: Ilenia
authors.initials: I
authors.lastname: Pennini
authors.forename: Loredana
authors.initials: L
authors.lastname: Vizzini
authors.forename: Franco
authors.initials: F
authors.lastname: Merletti
authors.forename: Anna
authors.initials: A
authors.lastname: Gillio-Tos
mesh_terms: D000328:Adult; D000368:Aged; D017931:DNA Primers / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D008875:Middle Aged; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: D017931:DNA Primers; D004279:DNA, Viral