High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR

Clin Chem Lab Med. 2011 Dec 20;50(4):655-61. doi: 10.1515/cclm.2011.835.

Abstract

Background: The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies.

Methods: The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and β-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67).

Results: An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed.

Conclusions: The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.

MeSH terms

  • DNA, Viral / analysis
  • DNA, Viral / genetics
  • Female
  • Genotyping Techniques / methods*
  • Humans
  • Papillomaviridae / genetics*
  • Papillomaviridae / isolation & purification*
  • Papillomaviridae / physiology
  • Real-Time Polymerase Chain Reaction / methods*
  • Viral Load

Substances

  • DNA, Viral