Detection and genotyping of human papillomavirus by real-time PCR assay.

BACKGROUND

Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients.

OBJECTIVE

Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples.

STUDY DESIGN

Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity.

RESULTS

Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10(3)cells using in-house HPV (6, 11 and 16) viral load assays.

CONCLUSION

The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.