Detection of human immunodeficiency virus type 1 DNA in dried blood spots by a duplex real-time PCR assay.
A dried blood spot (DBS) is a well-accepted means for the collection, transport, and storage of blood samples for various epidemiologic, serologic, and molecular assays for human immunodeficiency virus (HIV) studies. It is particularly important for mother-to-infant-transmission studies of affected individuals living in remote areas. We have developed a real-time PCR method to detect HIV type 1 (HIV-1) DNA in dried blood spots. A cellular gene, RNase P, was coamplified with the HIV-1 DNA in the same tube to monitor the DNA extraction efficiency and the overall assay performance. Our assay is a one-tube, single-step closed-system assay and uses a dUTP/uracil DNA glycosidase anti-PCR contamination control. The HIV-1 primers and probe were derived from a conserved region of the long terminal repeat. The detection of RNase P is attenuated by lowering the forward and reverse primer concentrations so that its amplification will not overwhelm the HIV-1 amplification and yet will provide a semiquantitative measurement of the quality of the isolated DBS DNA. We examined 103 HIV-1-seropositive and 56 seronegative U.S. adults and found that our assay has a sensitivity of 98.1% (95% confidence interval [CI], 95.5% to 100%) and specificity of 100% (95% CI, 99% to 100%). The positive and negative predictive values are 100% and 96.6%, respectively. This duplex PCR assay may be useful in identifying HIV-1-infected persons, particularly infants born to seropositive mothers in remote areas of the world.
Luo
Wei
W
National Center of HIV, STD and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Yang
Hua
H
Rathbun
Kimberly
K
Pau
Chou-Pong
CP
Ou
Chin-Yih
CY
D000328:Adult; D001800:Blood Specimen Collection / Q000379:methods*; D004247:DNA / Q000032:analysis; D004279:DNA, Viral / Q000097:blood*; D006679:HIV Seropositivity; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D043262:Ribonuclease P / Q000235:genetics; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D004247:DNA; D043262:Ribonuclease P