Abstract:
PCR provides one measure of proviral load in HIV-infected patients. Results are frequently and conveniently expressed as copy number where the patient sample PCR signal is measured against and interpreted through signals from PCR of standard concentrations of control proviral genomes. Copy number calculations provide an image of the intensity of infection. They are appealing in their simplicity but potentially misleading, since genetic polymorphism within and between patients (a hallmark of HIV infection) complicate copy number calculations. To address this issue we isolated DNA from three HIV-1 strains (IIIB, RF, and MN) and DNA from eight HIV+ patients (CD4 range 20 to 280 cells/microliters). We amplified this material with three primer pairs spanning two regions: SK38/39(gag); SK68/69(env); SK68-i/69-i(env-i aligned nearly at env but including degeneracies and inosine). Both env and env-i primers amplify IIIB and RF DNA, but only env-i primers amplify MN proviral DNA sequences owing to a single 3' mismatch against env; primers in gag amplify the three strains uniformly. HIV sequences from 7 of 8 patients are amplified nearly equivalently by env and by env-i primers, while the eighth is dramatically favored by env-i. IIIB-based copy number calculations on patient samples using amplification from either env or env-i yielded higher estimates than those based on gag primers. Primer pair, reference standard genotype, and patient HIV targets interact in PCR dynamics and copy number calculations. Longitudinal studies of relative HIV load for single patients is more straightforward, while cross sectional studies, defining copy number and potentially targeting different HIV genotypes, requires extensive analysis.
Keywords: DNA, Viral/GENETICS/*ISOLATION & PURIF Genes, env Genes, gag Genotype Human HIV/GENETICS/*ISOLATION & PURIF HIV Seropositivity/*MICROBIOLOGY Polymerase Chain Reaction/*METHODS ABSTRACT 930930
M9391183
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