GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus.
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.
de Araujo
Melissa Rodrigues
MR
Bauru School of Dentistry, University of São Paulo, Al. Octávio Pinheiro Brisola, 9-75, Bauru, São Paulo 17012-901, Brazil.
De Marco
Laura
L
Santos
Carlos F
CF
Rubira-Bullen
Izabel Regina Fisher
IR
Ronco
Guglielmo
G
Pennini
Ilenia
I
Vizzini
Loredana
L
Merletti
Franco
F
Gillio-Tos
Anna
A
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
D017931:DNA Primers; D004279:DNA, Viral