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.
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.
Authors: Emmanouela Sarchianaki; Stavros P Derdas; Markos Ntaoukakis; Elena Vakonaki; Eleni D Lagoudaki; Ismini Lasithiotaki; Anna Sarchianaki; Anastasios Koutsopoulos; Emmanouil K Symvoulakis; Demetrios A Spandidos; Katerina M Antoniou; George Sourvinos Journal: Tumour Biol Date: 2013-12-06
Authors: John Charles Rotondo; Lucia Oton-Gonzalez; Chiara Mazziotta; Carmen Lanzillotti; Maria Rosa Iaquinta; Mauro Tognon; Fernanda Martini Journal: Front Microbiol Date: 2020-11-19 Impact factor: 5.640