Detecting episomal or integrated human papillomavirus 16 DNA using an exonuclease V-qPCR-based assay

Virology. 2019 Nov:537:149-156. doi: 10.1016/j.virol.2019.08.021. Epub 2019 Aug 22.

Abstract

Screening for human papillomavirus (HPV) integration into host cell chromosomes typically requires large amounts of time and reagents. We developed a rapid and sensitive assay based on exonuclease V (ExoV) and quantitative polymerase chain reaction (qPCR) to determine HPV genome configurations in cell lines and tissues. We established the assay using genomic DNA from cell lines known to harbor integrated or episomal HPV16. DNA was incubated with ExoV, which is specific for linear DNA, and the DNA fraction resistant to digestion was measured by qPCR. The percent of DNA resistant to ExoV digestion was calculated relative to undigested DNA for determination of episomal or integrated HPV16. The ExoV assay was accurate, capable of distinguishing episomal from integrated HPV16 in cell lines and tissues. Future applications of the ExoV assay may include screening of HPV genome configurations in the progression of HPV-associated cancers.

Keywords: Episomal; Exonuclease V; HPV16; Integrated; qPCR.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • DNA, Viral / analysis*
  • DNA, Viral / genetics
  • Exodeoxyribonuclease V / metabolism*
  • Human papillomavirus 16 / genetics*
  • Human papillomavirus 16 / growth & development
  • Humans
  • Plasmids*
  • Proviruses / genetics*
  • Real-Time Polymerase Chain Reaction / methods*
  • Virus Integration*

Substances

  • DNA, Viral
  • Exodeoxyribonuclease V