Susceptibility testing by polymerase chain reaction DNA quantitation: a method to measure drug resistance of human immunodeficiency virus type 1 isolates.

Abstract

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2-or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7-or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Antibody-dependent cellular cytotoxicity and neutralization of human immunodeficiency virus type 1 by high affinity cross-linking of gp41 to human macrophage Fc IgG receptor using bispecific antibody

“…For the PCR assay, in order to remove any contamination by HIV DNA, the virus stock was treated prior to infection with RNasefree DNase (Promega), at 10 U/ml and MgCI 2 at 1 mM for 45 min at 25 °C. These conditions have been shown not to affect infectivity as reported elsewhere (Eron et al, 1992) since viral production in HIV-1/DAS-infected peripheral blood lymphocytes is identical with or without pretreatment with RNase-free DNase (data not shown). Heat-inactivated virus (56 °C for 45 min), pretreated with RNase-free DNase was used to check whether our virus stock gave a rise in the PCR background level.…”

The Polymerase Chain Reaction and Plant Disease Diagnosis

“…Quantitative PCR will likely be helpful for determining what stage of pathogenesis is inhibited in other resistant host cultivars. Recently, quantitative PCR has also been used to measure the resistance of human immunodeficiency virus to antiviral drugs (47), and similar drug susceptibility testing of phytopathogens is feasible.…”

Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates

“…Coculture of infected PBLs with donor seronegative PBLs is required to generate virus stocks for use in the PBL assay, which has also been shown to select for subpopulations of HIV-1 variants (21,49). Assays that avoid extensive cocultivation have been proposed (10,35).…”