AEGiS-11IAC: A competitive PCR method for the quantification of HIV-2 proviral load.

11th International AIDS Conference

Vancouver, British Columbia — July 7-12, 1996

A competitive PCR method for the quantification of HIV-2 proviral load.

Int Conf AIDS 1996 Jul 7-12; 11:16 (abstract no. We.A.521)
Gomes P, Taveira N, Moniz-Pereira J, Santos-Ferreira MO, Lourenco MH; Dep. Microbiology, University of Lisbon, Lisbon, Portugal. Fax: 351.1.7934212.

OBJECTIVE: Development of a quantitative competitive polymerase chain reaction (cPCR) with ELISA detection of amplified products to quantify HIV-2 proviral load.

METHODS: For the quantification of the HIV-2 proviral load we built an internal standard (pPG) which has almost the same size and the same primer recognition sites of the target gene and, consequently, the same behaviour during PCR cycling. This seems to guarantee the independence of the results from any predictable or unpredictable variable that can affect amplification. Increasing known copy numbers of competitive template were added to replicate portions of the test specimen. DNA was amplified with 5'-biotin and 3' primers. The amplification product was captured on extravidin-coated microplates and quantified by hybridization with digoxigenin-labelled internal oligonucleotide probes. After revelation with an anti-digoxigenin alkaline phosphatase coupled antibody (anti-dig-AP), the amount of hybridization probe was determined by optical reading. Samples were quantified by spectrophotometric analyses. The regression curves for the samples were calculated by plotting the OD pPG/OD wild-type against pPG copy number. The copy number of the wild-type was calculated from the curve expression for ODpPG/ODwild-type=1.

RESULTS: PBMC viral load of 24 samples from 14 asymptomatic HIV-2 individuals detected by our method ranged from 1.5 to 80 copies per 103 PBMC. All samples had CD4+ cell counts greater than 500/mm3 except 7 that had CD4+ cell counts less than 500/mm3. Cellular virus load was also determined using D. Ho's method; only 9 (37%) samples were positive by this method.

CONCLUSIONS: Our method can provide meaningful measurements of HIV-2 proviral levels in individuals with high CD4 counts, in whom virus isolation tends to be difficult. Viral DNA load in PBMC varies widely among HIV-2 asymptomatic individuals and no correlation was found between HIV-2 copy numbers and CD4+ cell counts. This method is specific, nonisotopic, sensitive, quantitative and practical.

Keywords: AEGIS, HIV-2, Polymerase Chain Reaction, CD4 Lymphocyte Count, Viral Load, Digoxigenin, Oligonucleotide Probes, Templates, Genetic, DNA, Enzyme-Linked Immunosorbent Assay, genetics, ICA11

960707
WeA521