Quantitation of HIV-RNA by one-step RT real-time polymerase chain reaction (PCR)

Abstract

The quantitative evaluation of HIV-1 viral load in plasma is a pivotal marker for the diagnosis and prognosis of HIV-1 infection. In addition, the viral load yields basis information for therapy monitoring allowing a concrete analysis of treatment failure caused by emerging resistance to a specific antiretroviral compound. For these reasons, studies on molecular procedures to determine viral load quantitatively have increased in recent years. Approaches such as The HIV-1 Quantiplex Assay, The AMPLICOR HIV-1 MORNITOR Assay และ The NucliSens HIV-1 Assay are employed widely as reference techniques for diagnostic laboratories. But the cost of the tests still remains expensive (1,500 - 2,000 bath).For this reason the One-Step RT Real-Time PCR was developed and the cost of this One-Step RT Real-Time PCR is less expensive (1,000 bath). In house One-Step RT Real-Time PCR was compared with The Bayer. HIV-1 RNA 3.0 Assay (bDNA) reference standard technique for the quantitative determination of HIV-1 viral load. Our approach presented a good correlation between two methods (r = 0.787). To evaulate the reproducibility of assay, the intra-inter-assay coefficient of variation (CV) were determined. The %cv was low, <7% for both intra-inter assays, indicating that our assay has a high reproducibility. The specificity is 90.62%. The One-Step RT Real-Time PCR assay detected in 66 out of 73 as sensitivity is 90.41%. The accuracy of the in house the One-Step RT Real-Time PCR method is 90.47%. In conclusion, our in-house One-Step RT Real-Time PCR assay has a good correlation, reproducible, practical, high sensitive and cost-effective and will be even more improved in the future studies.