Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

Abstract

BackgroundHuman 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.ResultsA 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%).ConclusionThere 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.

Presence of human papillomavirus in semen of healthy men is firmly associated with HPV infections of the penile epithelium

“…Therefore, combined use of these assays may allow a semiquantitative impression of viral loads in these male samples, in which SPF10-PCR-DEIA positivity but GP5þ/6þ PCR-EIA negativity is indicative of low viral loads (<250 viral copies per sample), and double-positivity is indicative of moderate/ high viral loads (>250 viral copies per sample) (18). All HPV16-and/or HPV18-positive samples by GP5þ/6þ PCR and/or SPF10-PCR were subjected to additional quantitative type-specific real-time PCR using a method adapted from Seaman et al (19) (King et al, personal communication).…”

Low Prevalence of HPV in Male Sexual Partners of HR-HPV Infected Females and Low Concordance of Viral Types in Couples in Eastern Guangdong

“…Each reaction was comprised of 1× Roche Lightcycler TaqMan master mix, HPV specific primer pairs and fluorescentlytagged probes for HPV 6/11 or 16/18 in a total reaction volume of 10 μl (Seaman et al, 2010). Thermal cycle conditions consisted of an initial denaturation at 95 °C for 10 min, followed by 40 cycles of alternating 95 °C for 15 s and 60 °C for 30 s. For standard curves, real-time PCR was performed on a 10-fold dilution series of each purified plasmid containing a type-specific L1 amplicon ranging from 2 × 10 1 to 1×10 6 copies.…”

The Oral HIV/AIDS Research Alliance Program: lessons learned and future directions

“…Since then, developmental activities in the laboratory have included further development and implementation of quantitative oral HPV detection methods that have been used throughout the HPV-related OHARA trials (Seaman et al, 2010). Consistent with the OHARA goals, we assessed HIV-associated salivary gland disease (HIVSGD) and have detected and characterized HIVSGD-associated BKPyV, a polyomavirus, and putative HIVSGD etiologic agent.…”