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Published on: March 8, 2012
GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus
Melissa Rodrigues de Araujo1, Laura De Marco, Carlos F Santos
1Bauru School of Dentistry, University of São Paulo, Al. Octávio Pinheiro Brisola, 9-75, Bauru, São Paulo 17012-901, Brazil.
Background:
Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions.
Objectives:
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.
Study Design:
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.
