Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR

Makiko Kondo, Koji Sudo, Rie Tanaka, Takako Sano, Hiroko Sagara, Shinya Iwamuro, Yutaka Takebe, Mitsunobu Imai, Shingo Kato

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14 Citations (Scopus)

Abstract

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 0.5 μg 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 (R2 = 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.

Original languageEnglish
Pages (from-to)141-146
Number of pages6
JournalJournal of Virological Methods
Volume157
Issue number2
DOIs
Publication statusPublished - 2009 May 1

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Keywords

  • Degenerate primer
  • HIV-1 DNA quantitation
  • Real-time PCR

ASJC Scopus subject areas

  • Virology

Cite this

Kondo, M., Sudo, K., Tanaka, R., Sano, T., Sagara, H., Iwamuro, S., Takebe, Y., Imai, M., & Kato, S. (2009). Quantitation of HIV-1 group M proviral DNA using TaqMan MGB real-time PCR. Journal of Virological Methods, 157(2), 141-146. https://doi.org/10.1016/j.jviromet.2008.12.006