Quantitative assay for group M (subtype A-H) and group OHIV-1 RNA detection in plasma

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the am plification and during the hybridization step. Sample viral load determinat ion was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curv e performed on known and increasing amounts of the reference HIV-1 (Ref HIV -1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-I strains belonging to t he different subtypes from group M, but also group O. were all detected. Ab solute quantification of purified HIV-I RNA copies gave identical results a s the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensi tivity. efficacy of material recovery, reproducibility and convenience of s ample handling. The microplate format of the assay combined with the colori metric detection provides a convenient tool and fulfills the requirement fo r routine molecular diagnostic laboratories. (C) 2001 Elsevier Science B.V. All rights reserved.