Alu-LTR real-time nested PCR assay for quantifying integrated HIV-1 DNA.
An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step real-time PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies. This Alu-LTR-based real-time nested PCR assay may be particularly useful to quantify integrated HIV-1 DNA in patients. It may also allow for the precise study of integration of HIV-1 DNA or HIV-1 based lentiviral vectors and may be a valuable tool to test future inhibitors of integration.
Brussel
Audrey
A
Département des Maladies Infectieuses, Institut Cochin, INSERM U567, CNRS UMR8104, Université René Descartes, Paris, France.
Delelis
Olivier
O
Sonigo
Pierre
P
D020087:Alu Elements; D004279:DNA, Viral / Q000032:analysis*; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009355:Neomycin; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics
D004279:DNA, Viral; D009355:Neomycin