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

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

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 ce...

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Bibliographic Details
Published in:Virology (New York, N.Y.) Vol. 537; pp. 149 - 156
Main Authors: Myers, J.E., Guidry, J.T., Scott, M.L., Zwolinska, K., Raikhy, G., Prasai, K., Bienkowska-Haba, M., Bodily, J.M., Sapp, M.J., Scott, R.S.
Format: Journal Article
Language:English
Published: United States Elsevier Inc 01-11-2019
Subjects:
Cells, Cultured
DNA, Viral - analysis
DNA, Viral - genetics
Episomal
Exodeoxyribonuclease V - metabolism
Exonuclease V
HPV16
Human papillomavirus 16 - genetics
Human papillomavirus 16 - growth & development
Humans
Integrated
Plasmids
Proviruses - genetics
qPCR
Real-Time Polymerase Chain Reaction - methods
Virus Integration
Exonuclease V
qPCR
Integrated
Episomal
HPV16
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Summary: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. •The ExoV assay is rapid and sensitive in detecting episomal and integrated HPV DNA.•The ExoV assay with HPV copy number identified episomal from type 1 or 2 integrants.•The ExoV assay monitors the physical state of HPV16 in organotypic raft tissues.
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ISSN:0042-6822
1096-0341
DOI:10.1016/j.virol.2019.08.021