Methodology
Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR
1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA
2 Department of Dental Ecology, School of Dentistry University of North Carolina at Chapel Hill, NC, USA
3 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, NC, USA
4 Biostatistics Core Facility, University of North Carolina at Chapel Hill, NC, USA
5 College of Dental Medicine, Western University of Health Sciences, Pomona, CA, USA
6 Department of Medicine, The Miriam Hospital, Brown University, Providence, RI, USA
7 Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
Virology Journal 2010, 7:194 doi:10.1186/1743-422X-7-194
Published: 19 August 2010Abstract
Background
Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts.
Results
A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%).
Conclusion
There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis.
