title: Development and validation of a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in blood cells from subjects undergoing a 1-year immunotherapeutic treatment.
abstract: OBJECTIVE
  To validate a quantitative polymerase chain reaction method developed to measure HIV-1 DNA in peripheral blood mononuclear cells.
  
  
  METHODS
  The assay was used to measure HIV-1 DNA in 15 consecutive blood samples taken from subjects enrolled in a multicenter, randomly allocated, double-blind, placebo-controlled trial using an HIV-1 immunogen. The assay was validated following the United States Pharmacopeia guidelines. The analytical parameters assessed were sensitivity, specificity, linearity and precision.
  
  
  METHODS
  The quantitative analysis was obtained by (1) co-amplifying HIV-1 DNA targets with an endogenous control (globin); (2) extrapolating the target values using HIV-1 and globin standard curves; and (3) normalizing the HIV-1 copy numbers to the globin copy numbers (genomic DNA load).
  
  
  RESULTS
  With United States Pharmacopeia assay validation methodology, the HIV-1 DNA polymerase chain reaction assay proved to be sensitive, specific, linear and precise and the evaluation of the relative difference between two consecutive blood samples was reproducible. The intra-assay variability, which examines the reproducibility of replicates, was determined using a conservative assessment (tolerance intervals). We established that an increase of 60% or more in the number of DNA copies or a decrease of 38% or more was significantly greater than the variation due to random or experimental error and therefore attributed this variability to a significant change in the HIV-1 DNA copy number.
  
  
  CONCLUSIONS
  We developed and validated a polymerase chain reaction method for the precise quantitation of HIV-1 DNA in peripheral blood mononuclear cells. This assay was able to detect changes in viral loads in HIV-1-infected asymptomatic subjects enrolled in a double-blind placebo-controlled trial using an HIV-1 immunogen.
authors.affiliation: Immune Response Corporation, Carlsbad, California 92008.
authors.forename: F
authors.initials: F
authors.lastname: Ferre
authors.forename: A L
authors.initials: AL
authors.lastname: Marchese
authors.forename: S L
authors.initials: SL
authors.lastname: Griffin
authors.forename: A E
authors.initials: AE
authors.lastname: Daigle
authors.forename: S P
authors.initials: SP
authors.lastname: Richieri
authors.forename: F C
authors.initials: FC
authors.lastname: Jensen
authors.forename: D J
authors.initials: DJ
authors.lastname: Carlo
mesh_terms: D015415:Biomarkers / Q000097:blood; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics*; D004311:Double-Blind Method; D015658:HIV Infections / Q000097:blood / Q000382:microbiology* / Q000628:therapy*; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007167:Immunotherapy; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014766:Viremia / Q000097:blood / Q000382:microbiology / Q000628:therapy
chemical_list: D015415:Biomarkers; D004279:DNA, Viral