RESUMO
A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.
Assuntos
DNA Viral/análise , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , Ligação Competitiva , Sequência Consenso , DNA Viral/genética , Genes env , Deleção de Sequência , Moldes GenéticosRESUMO
A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.
Assuntos
DNA Viral/análise , HIV-1/genética , Reação em Cadeia da Polimerase , Provírus/genética , Ligação Competitiva , Primers do DNA , Reações Falso-Negativas , Genes gag/genética , Soropositividade para HIV/virologia , Humanos , Leucócitos Mononucleares/virologia , Plasmídeos , Controle de Qualidade , Moldes GenéticosRESUMO
Controversy exists as to whether treatment of HIV-1-seropositive hemophiliacs with blood coagulation products of high purity might help prevent the decline of CD4-positive lymphocytes and thus delay progression toward AIDS. As viral load has recently been shown to be associated with disease progression in HIV-1 infection, we tested for a possible direct interference of high- or intermediate-purity blood coagulation products with replication of HIV-1. The data obtained revealed comparable replication of HIV-1 in the presence and absence of all blood coagulation products tested (assessed by PCR-based quantitation of proviral HIV-1 DNA in infected cells after a 10-day incubation period). These data suggest that coagulation factor concentrates per se will also have no direct effect on HIV-1 replication in vivo.