Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR.
The level of human immunodeficiency virus type 1 (HIV-1) proviral DNA is likely to be an important marker of the long-term effectiveness of highly active antiretroviral therapy. A new method was developed for quantifying HIV-1 group M proviral DNA using TaqMan real-time PCR, in which degenerate primers and an MGB probe were used to resolve the difference in amplification efficiencies among different subtypes. The present assay provided good linearity and accuracy in the range of 4-5000 copies of proviral DNA in 0.5microg of cellular DNA. The intra-assay and inter-assay coefficients were <31.6% and <30.1%, respectively. In 19 HIV-1 clinical isolates of six subtypes (A, B, C, CRF01_AE, F, and G), quantitation values by the real-time PCR assay matched closely those by Poisson distribution analysis of PCR results at endpoint dilution (R(2)=0.988). This assay is characterized by the use of degenerate primers and having been validated by comparing with a Poisson distribution-based assay. The present real-time PCR assay is highly sensitive, linear, reproducible, accurate, and independent of group M subtypes. The assay will be useful for studying the relationship between HIV-1 proviral loads and the long-term efficacy of antiretroviral therapy for subtype B as well as non-B subtype strains.
Kondo
Makiko
M
Kanagawa Prefectural Institute of Public Health, Shimomachiya, Chigasaki, Japan. kondo.dvqy@pref.kanagawa.jp
Sudo
Koji
K
Tanaka
Rie
R
Sano
Takako
T
Sagara
Hiroko
H
Iwamuro
Shinya
S
Takebe
Yutaka
Y
Imai
Mitsunobu
M
Kato
Shingo
S
D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics*; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019562:Viral Load / Q000379:methods*
D017931:DNA Primers; D004279:DNA, Viral