Surveillance technology for HIV-1 subtype C in Ethiopia: An env-based NASBA molecular beacon assay to discriminate between subcluster C and C′

Forty-nine samples with known C2V3 sequences were used for the evaluation of an env-based molecular beacon assay to distinguish between the two genetic subclusters C and Cwhich characterize the HIV-1 epidemic in Ethiopia. Two subcluster C and two subcluster Cbeacons targeting two different loci in the C2V3 region were developed. Using a three beacon-based (2C and 1C � = C prime), isothermal amplification assay, concordance with DNA sequencing was achieved for 43 (87.8%) samples. Sensitivity was 81.8% and specificity 97.4% for subcluster C beacons. For the subcluster Cbeacon, a sensitivity of 97% and a specificity of 87.5% was achieved. Five samples were ambiguous by sequencing of which two samples were subcluster Cby the beacon assay and one subcluster C. Two of the samples remained ambiguous with different beacon-pair combinations as well. From samples with a clear C or Cphylogeny by sequencing, three were undetected by the first-line beacon genotyping assay. Genotype ambiguity was resolved in the three samples using beacon pair combinations restricted to each targeted locus. The beacons were evaluated further in a panel including all HIV-1 subtypes. Four of five subtype C isolates were identified correctly, and no cross-reactivity was observed with other subtypes. © 2005 Elsevier B.V. All rights reserved.