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.
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.
Ferre
F
F
Immune Response Corporation, Carlsbad, California 92008.
Marchese
A L
AL
Griffin
S L
SL
Daigle
A E
AE
Richieri
S P
SP
Jensen
F C
FC
Carlo
D J
DJ
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
D015415:Biomarkers; D004279:DNA, Viral