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. 1999 Jul;73(7):6099-103.
doi: 10.1128/JVI.73.7.6099-6103.1999.

Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy

S R Lewin  1 , M Vesanen, L Kostrikis, A Hurley, M Duran, L Zhang, D D Ho, M Markowitz
Affiliations

Use of real-time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1-infected individuals on prolonged effective antiretroviral therapy

S R Lewin et al. J Virol. 1999 Jul.

Abstract

We have designed a novel, precise, and sensitive assay to measure unspliced (US) human immunodeficiency virus type 1 (HIV-1) mRNA in peripheral blood mononuclear cells of HIV-1-infected individuals by using real-time PCR and molecular beacons. Individuals were classified as either well suppressed (WS) or partially suppressed, based on longitudinal measurements of plasma HIV-1 RNA. The proportion of individuals with US mRNA undetectable over time was significantly higher among WS individuals; however, 30% of WS subjects still had detectable US mRNA after 24 months of effective antiviral therapy.

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Figures

FIG. 1
FIG. 1
(A) Location of primers and beacon on the HIV-1 genome (adapted from reference 22). The primers and beacon shown correspond to the following positions on pNL4-3: H13, nucleotides (nt) 626 to 648; H4, nt 879 to 901; SL30, nt 641 to 865; H10, nt 6070 to 6093; H2, nt 8504 to 8526; and H1, nt 6080 to 6102. (B) Real-time PCR quantification of HIV-1 US mRNA. A reading of change in fluorescence (Rn) as a function of cycle number is demonstrated for a range of known input copy numbers of in vitro-generated HIV-1 US mRNA transcripts. The copy numbers range from 50,000 to 50 copy equivalents per reaction. The cycle number at which the mean fluorescence rises 10 standard deviations above the baseline is called the threshold cycle (CT). The CT is shown for duplicates of the standards used. (C) Relationship of known number of HIV-1 transcripts to the CT. The CT is directly proportional to the log of the input copy equivalents, as demonstrated by the standard curve generated.
FIG. 1
FIG. 1
(A) Location of primers and beacon on the HIV-1 genome (adapted from reference 22). The primers and beacon shown correspond to the following positions on pNL4-3: H13, nucleotides (nt) 626 to 648; H4, nt 879 to 901; SL30, nt 641 to 865; H10, nt 6070 to 6093; H2, nt 8504 to 8526; and H1, nt 6080 to 6102. (B) Real-time PCR quantification of HIV-1 US mRNA. A reading of change in fluorescence (Rn) as a function of cycle number is demonstrated for a range of known input copy numbers of in vitro-generated HIV-1 US mRNA transcripts. The copy numbers range from 50,000 to 50 copy equivalents per reaction. The cycle number at which the mean fluorescence rises 10 standard deviations above the baseline is called the threshold cycle (CT). The CT is shown for duplicates of the standards used. (C) Relationship of known number of HIV-1 transcripts to the CT. The CT is directly proportional to the log of the input copy equivalents, as demonstrated by the standard curve generated.
FIG. 1
FIG. 1
(A) Location of primers and beacon on the HIV-1 genome (adapted from reference 22). The primers and beacon shown correspond to the following positions on pNL4-3: H13, nucleotides (nt) 626 to 648; H4, nt 879 to 901; SL30, nt 641 to 865; H10, nt 6070 to 6093; H2, nt 8504 to 8526; and H1, nt 6080 to 6102. (B) Real-time PCR quantification of HIV-1 US mRNA. A reading of change in fluorescence (Rn) as a function of cycle number is demonstrated for a range of known input copy numbers of in vitro-generated HIV-1 US mRNA transcripts. The copy numbers range from 50,000 to 50 copy equivalents per reaction. The cycle number at which the mean fluorescence rises 10 standard deviations above the baseline is called the threshold cycle (CT). The CT is shown for duplicates of the standards used. (C) Relationship of known number of HIV-1 transcripts to the CT. The CT is directly proportional to the log of the input copy equivalents, as demonstrated by the standard curve generated.
FIG. 2
FIG. 2
Cross-sectional analysis of US mRNA copies per microgram of mRNA in WS and PS individuals. Levels of US mRNA in PBMC from WS and PS individuals (circles) and median values (black lines) are shown. Data from three nonsuppressed individuals (diamonds) are shown as positive controls. There is a significant difference between WS and PS individuals in the levels of US mRNA at 12 months (P = 0.03) and 24 months (P = 0.11). The lower limit of detection of US mRNA was 50 copies/μg of total RNA.
FIG. 3
FIG. 3
Sequential changes in plasma viral load (squares in top row), CD4 lymphocyte count (circles), and US mRNA level (squares in bottom row) after combination therapy. The infected individual’s designation (e.g., 2003), the treatment regimen (AZT, zidovudine; 3TC, lamivudine; RIT, ritonavir; SQV, saquinavir; IDV, indinavir), and the classification of suppression are listed at the top of each panel. The lower limits of detection of plasma viral load and US mRNA were 50 copies/ml and 50 copies/μg of total RNA, respectively. NS, not suppressed.
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