Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by the TaqMan Real-Time PCR Assay

A variety of real-time PCR assays have been applied for quantification of human immunodeficiency virus type 1 (HIV-1) (4-7). Recently, Desire et al. have reported a TaqMan PCR for quantification of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) (3). This method targets the polymerase (pol) gene of HIV-1 subtype B. Based on their data, Desire et al. have concluded that HIV-1 proviral DNA load in PBMCs does not correlate with HIV-1 RNA level in plasma and CD4+ lymphocyte counts. This conclusion is contradictory to those previously drawn by us (2) and recently drawn by others (8). In order to clarify if the use of different DNA quantification methods may have contributed to this controversy, we decided to compare the TaqMan PCR by Desire et al. with a TaqMan PCR recently developed in our laboratory. Our TaqMan PCR targeted the long terminal repeat region (LTR) of the HIV-1 major group. The sequences of the sense and antisense primers were 5′-GCCTCAATAAAGCTTGCCTTGA-3′ and 5′-GGGCGCCACTGCTAGAGA-3′. The probe sequence was 5′-CCAGAGTCACACAACAGACGGGCACA-3′. The 5′ and 3′ ends of the probe were labeled with FAM and TAMRA dyes. The sensitivity of the assay was estimated to be five copies of HIV-1 plasmid DNA (Applied Biosystems). We randomly selected 25 PBMC samples, which had been earlier confirmed to be HIV-1 DNA positive by a nested PCR (1). The HIV-1 DNA was extracted from the PBMCs with the Qiagen blood kit. Five microliters of each DNA extract (equivalent to 2 × 10 5 PBMCs) was tested with both the pol- and LTR-based TaqMan PCR assays on the same plate for 2 cycles) were obtained by the pol TaqMan PCR (Table ​(Table1).1). Furthermore, HIV-1 DNA in seven HIV-1-positive PBMC samples was not detected by the pol TaqMan assay. Among those seven HIV-1 false-negative samples, three were PBMC culture positive for HIV-1. Our results suggest that the pol TaqMan PCR exhibits an unsatisfactory low sensitivity on the selected sample panel. The fact that some of the tested samples belonged to a subtype other than B may partly contribute to the high false negativity of the HIV-1 pol TaqMan PCR assay. However, the lower sensitivity was also seen when HIV-1 subtype B SF2 strain was tested with the pol TaqMan PCR (Table ​(Table1).1). Therefore, we further examined the pol TaqMan PCR design by using PrimerExpress software (version 2.0). The melting temperature of the reverse primer P2 was significantly lower than that of the forward primer P1 (50.4 versus 60.5°C). At the reported annealing temperature (60°C), an asymmetric amplification could have been taking place during PCR due to the poor hybridization of the P2 primer with the template, resulting in decreased amplification efficiency. Considering that HIV-1 load turns out to be extremely low after efficient antiretroviral therapy (ART), we believe that a highly sensitive real-time PCR is demanded for the purpose of ART monitoring. TABLE 1. Comparison of the numbers of Ct obtained by two TaqMan PCRs for HIV-1 DNA detection in PBMCs