Establishment of an end-point limiting-dilution PCR assay for detection of human immunodeficiency virus type 1 quasispecies

Abstract

Objective To establish a novel end-point limiting-dilution PCR (EPLD-PCR) assay to detect human immunodeficieney virus type 1 (HIV-1) quasispecies and evaluate its application value. Methods The multiplex limiting-dilution nested PCR base on the second round TaqMan real time PCR ( N-RT PCR) and regular PCR, end-point PCR and sequencing technology were used for detection of the sequences of HIV-1 quasispecies env gene c2-v3-c3 region and gag gene p17 region from the PBMCs of 3 HIV-1 infected patients with low level viral load. All sequences of c2-v3-c3 and p17 were confirmed by sequencing. Phylogenetic tree analysis for nucleetide sequences and alignment analysis for amino acid sequences were performed to evaluate the result of HIV-1 quasispecies assay. The HIV-1 DNA copy number in per million PBMCs was calculated by QUALITY program. Results Total 1 207 N-RT PCRs were carried out for all 3 patients' samples from different visiting time points. Sixty-five sequences of c2-v3-c3 and 101 sequences of p17 were obtained from regular nested second round PCRs; the quantification of HIV-1 DNA in per million PBMCs from 3 patients was between 1.34 copies to 53.76 copies. Phylogenetic tree analysis for all nucleotide sequences showed that the sequences in different patients were separated and they got together specially in the same patient. Amino acid sequences alignment analysis showed that HIV-1 quasispecies sequences were simplex at early stage of infection and became more complex alone with increase of infected time. Conclusions The end-point limiting-dilution real-time PCR technology described in this study is a novel, sensitive, specific and high throughput PCR assay to detect human immunedeficiency virus type 1 quasispecies. This PCR assay could be applied for detection of HIV-1 quasispecies in HIV-1 infected populations with low level viral load. Key words: HIV-1 ; Polymerase chain reaction