Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types

Abstract

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra-and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intraassay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Among all types of human papillomavirus (ϳ100 types) the ones classified as oncogenic types are of clinical significance due to their association with cervical neoplastic development and carcinoma. Although most HPV infections are transient, a number of host and viral factors may lead to a persistent infection thus increasing the risk of developing neoplasia.1 Understanding the natural history of oncogenic HPV infection and the predictive value of one or several HPV test, is, therefore, essential for the development of appropriate cervical cancer prevention and control programs. The limitations of cytological analysis have pressured the development of alternative screening methods in population studies, with molecular diagnosis emerging as a promising diagnostic tool in cervical cancer prevention.2 Quality assurance of these emerging technologies is paramount for large-scale clinical investigations.3 In population-based studies, questions of reliability, sensitivity, specificity, reproducibility, and scalable capacity for automation are central in selecting assays to determine factors associated with cervical carcinogenesis.Prevalence studies conducted worldwide indicate that approximately 30% of women are infected with HPV. Therefore, the use of HPV testing as a primarily screening tool is not sufficient. 4 Alternative measures that may have greater predicted value, such as HPV viral load, are currently under investigation. Several studies have correlated HPV viral load and its prognostic significance in the evolution of cervical intraepithelial neoplasia. 5,6 The methodologies used to quantitate viral burden have varying levels of specificity and sensitivity and performance evaluation is frequently not comprehensive. Hybrid capture has been a common technique used to determine HPV viral load, but does not have the capacity to diffe...

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma

“…29 No cross-reactivity was observed in any of the eight HPV genotypes. High intra-and inter-assay reproducibilities were also obtained (data not shown).…”

“…MGB probes were labeled with a corboxyfluorescein reporter dye at the 5 0 end and a nonfluorescent quencher at the 3 0 -end of probe. Primers described by Flores-Munguia et al 29 for the E6/E7 regions were used for HPV 16, 18, 31, 45, 52, and 58, whereas primers were designed in-house using Primer Express software (Applied Biosystems) for the E6/E7 regions of HPV types 33 and 35. For the HPV 16 integration assay, primers for HPV 16 E2 described by Peitsaro et al 34 were used.…”

Cross‐sectional analysis of oncogenic HPV viral load and cervical intraepithelial neoplasia

“…HPV viral load was determined based on HPV type-specific real-time PCR assays as described by Flores-Munguia et al 28 This Real-time PCR (RTPCR) method is based on the Taqman assay where the 5 0 exonuclease activity of Taq DNA polymerase is used to produce fluorescence that is measured during each cycle of the PCR reaction. Briefly, the E6/E7 regions of HPV types 16 (nt 65-857), 18 (nt 87-907), 31 (nt 39-856), 39 (nt 44-921), 45 (nt 75-906), 51 (nt 88-865), 52 (nt 93-852) and 58 (nt 77-869) were used as input for the RTPCR oligonucleotide design software program, Primer Express (V2.0, Applied Biosystems, Foster City, CA).…”

“…The primers and probe sequences for HPV16 and HPV18 were from Tucker et al 29 The nucleotide sequences of the selected HPV types (16,18,31,39,45, 51, 52 and 58) primers and probes sequences are described elsewhere. 28 The standard curves for absolute quantification of HPV type specific target transcripts were generated as previously reported 28 using the E6-E7 region of HPV16, HPV18, HPV45 and HPV51 and the complete sequence for HPV31, HPV39, HPV52 and HPV58, that had been cloned into plasmid vectors. Real-time PCR reactions were performed in a 10 ll volume using the ABI PRISM 1 7900HT instrument (Applied Biosystems, Foster City, CA).…”

Correlates of Human Papillomavirus Viral Load with Infection Site in Asymptomatic Men

“…Specimens from seven anatomic sites of those men who were HPV-16 -positive at one or more anatomic sites by the reverse line blot method were evaluated for HPV-16 viral load using a quantitative real-time PCR assay (TaqMan) as previously described (21). Briefly, the standard curve for absolute quantification of HPV-16 was generated using the E6-E7 region of HPV-16 that had been cloned into plasmid vectors (21). Real-time PCR reactions were done in a 20 AL volume using the ABI PRISM1 7900HT instrument (Applied Biosystems).…”