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Amel Jamehdar S, Sabahi F, Forouzandeh M, Haji Abdolbaghi M, Kazemnejad A, Edalat R, et al . Application of SYBR-Green Real Time RT-PCR for quantification of human immunodeficiency virus type-1 (HIV-1) and comparison with COBAS AMPLICOR test. mjms 2008; 10 :43-50
URL: http://mjms.modares.ac.ir/article-30-9162-en.html
Application of SYBR-Green Real Time RT-PCR for quantification of human immunodeficiency virus type-1 (HIV-1) and comparison with COBAS AMPLICOR test
Saeid Amel Jamehdar1 , Farzaneh Sabahi 1, Mahdi Forouzandeh2 , Mahboubeh Haji Abdolbaghi3 , Anooshiravan Kazemnejad4 , Rosita Edalat5 , Mehrnaz Rasoolinejad3 , Farzaneh Barkhordari6 , Fereidoun Mahboudi6
1- Department of Virology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2- Department of Biotechnology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3- Department of Infectious Diseases, AIDS Research Center of Iran, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
4- Department of Biostatistics, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
5- Department of Virology, Pasteur Institute of Iran, Tehran, Iran
6- Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Abstract:   (7877 Views)
Objective: In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed. Materials and Methods: This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26 seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIV-1 Monitor test. Results: The results demonstrated that this technique could detecte up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers (5×102-5×109). Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results (R2= 0.95). Conclusion: On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool of clinical outcome. Indeed, the development and stabilization of HIV-1 RNA assays have given physicians a unique tool for monitoring HIV-1 patients treated with antiviral drugs. In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV-1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test (5×10 9 versus 7.5×10 5). This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis. Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than
Keywords: Real Time RT-PCR, COBAS Amplicor Monitor Test, Real Time RT-PCR, COBAS Amplicor Monitor Test
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Received: 2010/01/26 | Accepted: 2007/08/28
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