Evaluation of a competitive quantitative RT-PCR-ELISA system for quantification of HIV-1 RNA in plasma: comparison with COBAS Amplicor HIV-1 monitor test
Abstract number: 1733_670
Amel jamehdar S., Sabahi F., Forouzandeh M., Haji Abdolbaghi M., Kazemnejad A., Mahboudi F., Edalat R., Ravanshad M.
Objectives: Quantification of the human immunodeficiency virus type 1 RNA levels (viral load) has become an indispensable tool for the assessment of patient prognosis and for the monitoring of antiretroviral therapy. The access to commercial assays for viral load monitoring in IRAN is still limited due to the high costs. The aim of this study was to design a competitive quantitative RT-PCR-ELISA system for sensitive amplification of HIV-1 pol gene and evaluation of the treatment of 80 HIV-1 seropositive patients. Results of this assay were compared with the COBAS Amplicor HIV-1 monitor test.
Methods: Whole blood samples from 80 HIV-1 seropositive and 40 seronegative blood donors were collected in vaccutainer tube. Plasma was separated and stored at -80°C. Total RNA was extracted according to the protocol supplied by the commercial viral RNA kit (Qiagen). A quantitative HIV-1 test is described based on competitive quantitative RT-PCR assay combine with hybridisation as a detection system. The internal RNA standard (IS) was designed by SOEing mechanism, specifically to be competitive during the amplification step. Sample viral load determination was carried out with one RT-PCR in the presence of 103 IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 RNA co-amplified with a constant amount of the IS RNA.
Results: The results demonstrated that this technique detected 5 HIV-1 RNA copies per milliliter of plasma. The assay had a linear range from 103 to 106 HIV-1 copies. A positive linear correlation between the two tests (r2 = 0.88) was found. Overall, no significant differences were found in plasma viral load quantitation between both assays, therefore this assay is suitable for viral load quantitation of HIV-1 plasma samples.
Conclusions: Our data demonstrates that our competitive quantitative RT-PCR-ELISA system is a fast and reliable method for the detection and quantitation of HIV-1 RNA and this technique could be applied during the monitoring of HAART treatment.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
|Back to top|