Background: The presence of 2-LTR circles in the PBMN cells of patients with <50 copies/mL of plasma HIV-1 RNA is indicative of ongoing viral replication. There is controversy about the half-life of these circles in primary cells. We have adapted the circle assay that was previously published for the ABI Prism 7700 so it can now be performed using a Roche LightCycler. Specific issues requiring protocol amendments were: DNA extraction; abstraction of PCR reagents onto the glass capillary surface; primer dimer formation, and circle half-life in primary cells.

Methods: 10 million PBMN cells frozen as pellets were thawed in DMSO. 2-LTR circles were separated from genomic DNA by proteinase K digestion followed by an alkali lysis mini-prep and exonuclease (Plasmid-Safe DNase) digestion.  LightCycler PCR was performed using a standard PCR mix (Qiagen “HotStar” or Platinum-Taq) modified by adding extra MgCl_2, and BSA or High Density Lipoprotein (HDL) or RNase A. Comparison was made with the Roche and BioGene LightCycler mixes. HPLC-purified primers (which included AA at the 3’ end) together with modified annealing and denaturation temperatures were also used. A plasmid containing the entire HIV-1 2-LTR sequence was used for quantification.  C8166 cells were infected with HIV-1 IIIB. PBMN cells and MDMs were infected with primary viral isolates. Antiretroviral drugs were added 2-4 days post infection and cells harvested for 2-LTR circles at regular intervals.

Results: Digestion with proteinase K followed by alkali-lysis mini-prep and post-purification digestion with exonuclease gave better results than the use of alkali lysis only. Using a spiked plasmid, >70% recovery of circles was achieved. A “homemade mix” or the Qiagen “HotStar” mix were as effective as expensive, specially formulated LightCycler PCR reagents. BSA, HDL or RNase significantly reduced abstraction of the PCR reagents onto the glass capillary surface. The addition of RNase to PCR mixes reduced primer dimer formation. The half-life of 2-LTR circles in T cell lines, PBMN cells and MDMs was 4-14 hours.

Conclusions: Several important protocol amendments were required to perform PCR for 2-LTR circles efficiently and effectively in the glass capillaries used in the Roche LightCycler. Using our new method, it is now possible to routinely detect down to 5 copies of 2-LTR circles per reaction. The short half-life of these circles in a variety of cells has been confirmed, and the results validate the use of this assay to monitor acute infection events in vitro and in vivo.