Competitive RT-PCR coupled with Enzyme-Linked Immunosorbent Assay for Quantification of Human Immunodeficiency Virus Type 1 RNA: Comparison with Commercial Quantitative Assay

دومين همايش ساليانه پژوهش ايدز در ايران

22 آبان 1387، تهران - ايران

Presentation Type: Speech

Background: Different techniques have been developed for the quantification of HIV RNA, including competitive reverse transcriptase PCR (RT- PCR), branched-DNA (b-DNA) signal amplification and nucleic acid sequence-based amplification (NASBA). Some of these assays are now commercially available. However, the access to these commercial assays for viral load monitoring in IRAN is still limited due to the high costs. We developed and evaluated a quantitative-competitive RT-PCR-enzyme-linked immunosorbent assay to sensitive measurement of HIV RNA levels in plasma. Results of this assay were compared with the COBAS Amplicor HIV-1 monitor test.
Material and Methods: The assay is based on co-amplification of an internal standard (IS) RNA and the wild-type HIV RNA of the sample. This internal RNA standard was designed by SOE mechanism, specifically to be competitive during the amplification step. A standard curve is stablished from parallel co-amplification of increasing amounts of external standard (ES) wild-type RNA and the same quantity of the IS. The RNA copy number is calculated from the signal ratio of wild-type to the IS. The accuracy of quantification was verified by comparison with reference commercial test, the Roche AMPLICOR HIV MONITOR assay.
Results: The results demonstrated that this technique detected 500 HIV-1 RNA copies per milliliter of plasma. The assay had a linear rang from 5 to 5×106 HIV-1 copies. Overall, no significant were found in plasma viral load quantitation between our assay and reference quantification method (R2=0.95, P<0.01). Therefore this assay is suitable for viral load quantitation of HIV-1 plasma samples.
Conclusion: The detection method must be taken into consideration as a part of the overall quantitative process. The standardization of routine procedures, such as microtiter plate-based hybridization assay, has considerably simplified this step of quantitation procedure. We have described a competitive RT-PCR procedure coupled to ELISA microtiter plate assay. For quantifying HIV RNA, Competitive RT-PCR seemed to be the method of choice. The method requires only one tube and includes a single control for RNA integrity and amplification efficiency.