Real-time PCR Assays Using Internal Controls for Quantitation of HPV-16 and Beta-Globin DNA in Cervicovaginal Lavages

J Virol Methods. 2003 Dec;114(2):135-44. doi: 10.1016/j.jviromet.2003.09.003.

Abstract

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cervix Uteri / virology*
  • DNA, Viral / analysis*
  • Female
  • Globins / genetics*
  • HeLa Cells
  • Humans
  • Papillomaviridae / genetics
  • Papillomaviridae / isolation & purification*
  • Papillomavirus Infections / virology
  • Polymerase Chain Reaction / methods
  • Polymerase Chain Reaction / standards*
  • Reproducibility of Results
  • Taq Polymerase
  • Uterine Cervical Neoplasms / virology
  • Vagina / virology*
  • Vaginal Douching
  • Viral Load

Substances

  • DNA, Viral
  • Globins
  • Taq Polymerase