Detection of phylogenetically diverse human immunodeficiency virus type 1 groups M and O from plasma by using highly sensitive and specific generic primers

Abstract

The high degree of genetic diversity within human immunodeficiency virus type 1 (HIV-1), which includes two major groups, M (major) and O (outlier), and various env subtypes within group M (subtypes A to J), has made designing assays that will detect all known HIV-1 strains difficult. We have developed a generic primer set based on the conserved immunodominant region of transmembrane protein gp41 that can reliably amplify as few as 10 copies/PCR of viral DNA from near-full-length clones representing group M subtypes A to H (subtypes I and J were not available). The assay is highly sensitive in detecting plasma viral RNA from HIV-1 strains of diverse geographic origins representing different subtypes of HIV-1 group M as well as HIV-1 group O. Of the 253 group M plasma specimens (subtypes A, 68 specimens; B, 71; C, 19; D, 27; E, 23; F, 33; and G, 12), 250 (98.8%) were amplified by using the gp41 M/O primer set. More importantly, all 32 (100%) group O plasma samples were also amplified with these primers. In vitro spiking experiments further revealed that the assay could reliably detect as few as 25 copies/ml of viral RNA and gave positive signals in HIV-1-seropositive specimens with plasma copy numbers below the limits of detection by all commercially available viral load assays. In addition, analysis of five seroconversion panels indicated that the assay is highly sensitive for early detection of plasma viremia during the "window period." Thus, the highly sensitive assay will be useful for early detection of HIV-1 in clinical specimens from all known HIV-1 infections, regardless of their genotypes and geographic origins.

FIG. 1

Sensitivity of gp41 M/O primers in detecting HIV-1 group M subtypes A to H near-full-length molecular reference clones. The known numbers of copies per PCR of the clones A (92UG037.1), B (YU2 [left set of lanes] and SG3 [right set of lanes]), C (92BR025.8), D (94UG114.1), A/E (90CF402.8), F (93BR020.1), A/G (92NG003.1), and H (90CF056.1) were amplified with gp41 M/O primers. For each subtype, lane 1 is 100 copies/PCR, lane 2 is 10 copies/PCR, lane 3 is 5 copies/PCR, lane 4 is 1 copy/PCR, and lane 5 is 0.1 copy/PCR.

FIG. 2

Sensitivity of gp41 M/O primers in detecting HIV-1 RNA in plasma with known copy numbers (A) and early seroconverters (B). (A) Normal plasma was spiked with known copy numbers of subtype B viruses prior to RNA extraction and amplification. (B) RNA extracts from longitudinal plasma specimens from five persons (panel no. 946 and 948 to 951) during the window period were amplified with gp41 M/O primers. The results with HIV-1 antibody (Ab), p24 antigen (Ag), and three commercial RNA tests (RNA) are shown as negative (−) and positive (+) at the bottom of each panel.