Quantification of HFV-integrated DNA in human cells by Alu-LTR real-time PCR.
Integration is described as a key step in viral replication of all retroviruses. A sensitive and quantitative measure of an integrated molecule is a good way to examine the importance of the integration step and to evaluate efficiency of retroviral vectors for gene transfer or anti-integrase drugs. Here, we report a sensitive and quantitative real-time polymerase chain reaction (PCR) technique to measure integrated viral DNA in human cells during a foamy virus (HFV) infection. This technique is based on two steps of PCR. The first round amplifies Alu-LTR (long terminal repeat) sequences resulting from viral integration. The second round of PCR is performed to quantify these events of integration. Quantification is monitored by the comparison of the amplification curve of the sample against a standard scale constituted of viral DNA from chronically infected cells. Sensitivity of this technique allows us to detect as few as 25 copies of HFV-integrated DNA in 50,000 cells.
Delelis
Olivier
O
Département des Maladies Infectieuses, Institut Cochin, INSERM 1567, CNRS UMR 81041, Université René Descartes, Paris, France.
Brussel
Audrey
A
Sonigo
Pierre
P
D020087:Alu Elements; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D005914:Globins / Q000032:analysis; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics; D016092:Spumavirus / Q000235:genetics*; D020079:Terminal Repeat Sequences; D016662:Virus Integration
D004279:DNA, Viral; D005914:Globins