Development and Validation of a HPV-32 Specific PCR Assay

Nicholas R Herrel,Nadia L Johnson,Jennifer E Cameron,Janet Leigh,Michael E Hagensee

doi:10.1186/1743-422x-6-90

Abstract

BackgroundHuman Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay.Materials and methodsAn HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects).ResultsThe HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard.ConclusionDue to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

Highlights

Human Papillomavirus-32 (HPV-32) has traditionally been associated with focalepithelial-hyperplasia (FEH)
The HPV-32 specific polymerase chain reaction (PCR) assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies
Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively

Summary

Introduction

Human Papillomavirus-32 (HPV-32) has traditionally been associated with focalepithelial-hyperplasia (FEH). This study utilized a complex series of PCR assays designed to detect a broad spectrum of HPV types including genital, oral, and cutaneous types found in humans as well as animal This was accomplished utilizing a series of primers for highly conserved sequences in the L1 open reading frame and radiolabeled probes for Southern blot analysis. In the advent of widespread use of highly active anti-retroviral therapy (HAART) an increase in oral warts has been observed while decreases in other types of oral lesions have been noted over the same time period [6-8] This coupled with the fact that most oral warts in HIV-positive individuals are due to HPV-32 makes further study into the natural history of these HPV-32 infections warranted

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