TABLE 21.

Comparison of rapid-cycle real-time PCR assays for the detection of HIV-1 and HIV-2 in clinical specimensa

HIV typeReferenceSpecimen type(s)Test platform/probe chemistryTargetSpecimen volume inputRange of quantitative resultsComparison to other assaysComments
HIV-1Brussel et al. 2003 (52)PBMC in whole bloodLightCycler/FRET hybridization probesOverlapping region between LTR and chromosomal Alu element in initial PCR; LTR gene in second round of PCR106 PBMC6 to 105 copies per 5 × 104 PBMCNo comparison with other assays.A nested PCR assay designed to quantify HIV-1 proviral DNA for in vitro infectivity experiments.
Candotti et al. 2004 (59)PlasmaStratagene Mx4000/TaqMan probe79-bp fragment in LTR region200 μl680 (95% detection rate) to 2 × 105 IU/mlCorrelated with single virus real-time PCR assay (R2 = 0.87) among 39 samples with HIV-1 RNA titers ranging from 200 to 2 × 105 IU/ml; 100% specificity among 266 healthy blood donor sera tested by anti-HIV-1/2 EIA.Intra- and interassay CV ranged from 1.6% to 3.4% and from 3.4 to 7%, respectively; equivalent detection of HIV-1 group M subtypes A to G; a multiplex assay which also detect HBV DNA and HCV RNA.
Desire et al. 2001 (93)PBMC in whole bloodABI Prism 7700/TaqMan probe199-bp fragment of pol gene5 × 106 PBMC5 to 105 copies per 106 PBMCNo correlation with HIV-1 RNA levels in 21 clinical plasma samples tested by Amplicor HIV-1 Monitor v1.5 or Quantiplex HIV RNA 2.0 assay.Quantify HIV-1 proviral DNA; intra- and interassay CVs were 13% and 27%, respectively.
Eriksson et al. 2003 (113)PBMC in whole bloodABI Prism 7700/TaqMan probe89-bp fragment of pol gene5 × 106 CD4+ cells2 to 105 copies per 104 CD4+ cellsNo comparison with other assays.Quantify HIV-1 proviral DNA; intra-assay %CV ranged from 30% to 34%
Ghosh et al. 2003 (140)Breast milkABI Prism 7700/TaqMan probeFragment of LTR gene1 ml102 to 107 copies/mlNo comparison with other assays.Quantify HIV-1 proviral DNA in breast milk.
Gibellini et al. 2004 (141)PlasmaLightCycler/SYBR Green142-bp fragment of gag gene1 ml50 to 5× 105 copies/mlDetected 4 (7%) more positive samples than Quantiplex HIV-1 RNA 3.0 bDNA assay (R2 = 0.91)Both intra- and interassay CVs were<4%.
among 56 plasma specimens from HIV-infected patients; 100% specificity on 25 samples from seronegative blood donors.
Gibellini et al. 2004 (142)PBMC in whole bloodLightCycler/SYBR Green266-bp fragment of pol gene5 × 106 PBMC5 to 104 copies per 106 PBMCNo correlation with HIV-1 RNA levels in 50 clinical plasma samples tested by Quantiplex HIV RNA 3.0 assay; 100% sensitivity and specificity on 50 HIV-1-seropositive and 20 seronegative clinical sera tested by conventional PCR assay.Quantify HIV-1 subtype B proviral DNA; intra- and interassay CVs were <5%.
Gueudin et al. 2003 (153)PlasmaLightCycler/TaqMan probe111-bp fragment of LTR gene200 μl1.5 × 103 to 5 × 105 copies/mlNo comparison with other assays.Specifically quantify HIV-1 group O RNA; both intra- and interassay CVs were<20%.
Ito et al. 2003 (197)Lymphoid tissue (tonsillar tissue)ABI Prism 7700/SYBR GreenFragments of various bp sizes in V3-V5 region of gp120 gene5 mg10 to 105 copies/reactionGood correlation with levels of HIV-1 p24 Ag in lymphoid tissue (R2 = 0.99).Quantify HIV-1 RNA in lymphoid tissue; both intra- and interassay CVs were <10%.
Palmer et al. 2003 (363)PlasmaABI Prism 7700/TaqMan probeFragment of gag gene1 to 7 ml102 to 108 copies/mlCorrelated with results of VERSANT HIV RNA 3.0 bDNA assay (R2 = 0.89) on 22 clinical specimens, but detected titers in 15 additional samples that were negative by bDNA test; 100% specificity on samples from seronegative blood donors.Intra-assay variability ranged from 13% to 37%.
Saha et al. 2001 (423)PlasmaLightCycler/scorpion probe104-bp fragment of pol gene?1 to 108 copies/reactionGood correlation with results of rapid PCR assay using molecular beacon probe (R2 = 0.99).Intra- and interassay variability ranged from 1% to 46% and from 5% to 20%, respectively.
Vet et al. 1999 (518)PlasmaABI Prism 7700/molecular beacon probeFragment of gag gene?10 to 106 copies/reactionCorrelated with results of Amplicor HIV-1 Monitor test v1.0.A multiplex assay which also quantifies HIV-1, HTLV-I, and HTLV-II RNA levels.
Victoria et al. 2003 (519)PBMC and lymphoid cell linesSmart Cycler/SYBR GreenFragment of LTR gene5 × 106 cells10 to 5 × 104 copies per 106 cellsNo comparison with other assays.Quanitfy HIV-1 RNA for in vitro infectivity experiments.
Weber et al. 2003 (529)Cell culture supernatantSmart Cycler/SYBR Green199-bp fragment of pol gene200μ l10 to 107 copies/reactionNo comparison with other assays.Quantify HIV-1 proviral DNA for in vitro infectivity experiments.
HIV-2Damond et al. 2001 (88)Whole bloodLightCycler/ TaqMan probe160-bp fragment of gag gene1 ml5 (100% detection rate) to 500,000 copies/105 PBMCHIV-2 proviral DNA levels correlated with positive cell cultures for HIV-2 virus (U = 26; P < 0.02) but not with rate of plasma HIV-2 RNA detection (U = 61; P< 0.07) among 29 HIV-2 antibody-positive patients.Requires nested RT-PCR and quantifies HIV-2 proviral DNA, with intra- and inter assay CVs ranging from 11.8% to 41% and 30% to 40%, respectively; equivalent detection of HIV-2 subtypes A and B.
Damond et al. 2002 (89)PlasmaLightCycler/ TaqMan probe89-bp fragment of gag gene1 ml250 to 5 × 105 copies/mlHIV-2 RNA detected at >250 copies/ml in 21 (54%) of 39 patients with detectable HIV-2 proviral DNA, with 100% specificity among 25 HIV-negative blood donors and 25 HIV-1 antibody-positive patients.Intra- and interassay CVs varied from 1% to 4.4%; equivalent detection of HIV-2 subtypes A and B.
Ruelle et al. 2004 (421)PlasmaLightCycler/SYBR Green I173-bp fragment in LTR region1 ml250 to 2.5× 107 copies/mlHIV-2 RNA detected at >250 copies/ml in 12 of 13 untreated HIV-2 antibody-positive patients but none in two HIV-1 antibody-positive and 1 HIV-negative sera.Intra- and interassay CVs varied from 0.2% to 5.6% and from 0.7% to 3.1%, respectively; equivalent detection of HIV-2 subtypes A and B.
Schutten et al. 2000 (442)PlasmaABI Prism 7700/TaqMan probe61-bp fragment of region between LTR and gag/pol ORF, or 67-bp fragment of gag gene400 μl500 (100% detection rate) to 5 × 106 copies/mlHIV-2 RNA detected in 13 (81%) of 16 HIV-2 antibody-positive (by Western blot) patients.Interassay CVs varied from 2% to 7.5%; differential quantification of HIV-2 subtype A and B RNAs.
Vet et al. 1999 (518)PlasmaABI Prism 7700/molecular beacon probeFragment of env gene?10 to 106 copies/reactionCorrelated with qualitative results of anti-HIV-2 antibody assay.A multiplex assay which also quantifies HIV-1, HTLV-I, and HTLV-II RNA levels.
  • a PBMC, peripheral blood mononuclear cells; LTR, long terminal repeat; EIA, enzyme immunoassay; CV, coefficient of variation; Ag, antigen; HTLV, human T-cell lymphotropic virus; RT, reverse transcription.