Development of a Novel Codon-Specific Polymerase Chain Reaction for the Detection of CXCR4-Utilizing HIV Type 1 Subtype B

Abstract

Insight concerning the switch in HIV-1 coreceptor use will lead to a better understanding of HIV-1 pathogenesis and host-virus dynamics. Predicting CXCR4 utilization by analyzing HIV-1 envelope consensus sequences is highly specific, but minority variants in the viral population are often missed resulting in low sensitivity. Commercial phenotypic assays are costly, and the development of sensitive in-house phenotypic assays to detect CXCR4-using HIV may not be feasible for some laboratories. A sensitive, inexpensive genotyping assay was developed to detect viral sequences associated with CXCR4-utilizing virus (X4). Codon-specific primer pairs were used to detect X4-associated codons at five positions in the HIV-1 envelope V3 loop (11, 13, 24, 25, and 32). Sixty plasma samples from HIV-1-infected individuals were analyzed by consensus sequencing and codonspecific PCR (CS-PCR). Forty-six of these were also phenotyped by Trofile or Enhanced Sensitivity Trofile (ESTA). CS-PCR detected X4 variants 17% more often than 11/24/25 consensus sequencing alone (n = 60), 30% more often than Trofile (n = 27), and in a limited data set, 16% more often than ESTA (n = 19). CS-PCR combined with consensus sequencing had approximately 80% concordance with ESTA.

Development and Validation of a Genotypic Assay to Quantify CXCR4- and CCR5-Tropic Human Immunodeficiency Virus Type-1 (HIV-1) Populations and a Comparison to Trofile®

“…A quantitative PCR (qPCR) amplified and assessed the total number of HIV RNA templates reverse transcribed into cDNA, with the forward primer annealing to the relatively conserved env C2 region and the reverse primer to the “ill.1” part of cDNA reverse transcription primer ( Table 1 ). Quantification standards used the env-containing plasmid p2-7 DNA [ 22 ]. The full volume of eluted cDNA (20 µL) was divided between four replicate 50 µL qPCR reactions using 0.75X SensiMix™ (Meridian Bioscience Inc., Cincinnati, OH, USA) and 0.3 µM primers (env6880F and ill.1; Table 1 ).…”