Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

Anny Armas Cayarga,Yaimé J González González,Yenitse Perea Hernández,Santiago Dueñas Carrera,Idania González Pérez,René Robaina Álvarez

doi:10.4061/2011/964831

Abstract

Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load.

Highlights

The quantitation of human immunodeficiency virus type1 (HIV-1) Ribonucleic acid (RNA) is a crucial factor to help clinicians decide whether to stop or continue antiretroviral combinations after switching or starting antiretroviral drug therapy [1]
In spite of the advantages that offer the use of the real-time positive control (PCR), which helps reducing the possibility of contamination, increasing the assay sensitivity, and improving the turnaround time [10], commercially available real-time PCR assays are just as expensive as the more standard nucleic acid viral load tests and rely on expensive, often dedicated equipment, that can only be used for these assays [10, 11]
A fragment of 120 bp located in the 5 long terminal repeat (LTR) region of the HIV-1 genome was amplified from RNA isolated from an HIV-1 subtypeB-infected serum, by reverse transcriptase-polymerase chain reaction (RT-PCR)

Summary

Introduction

The quantitation of human immunodeficiency virus type (HIV-1) RNA is a crucial factor to help clinicians decide whether to stop or continue antiretroviral combinations after switching or starting antiretroviral drug therapy [1]. In spite of the advantages that offer the use of the real-time PCR, which helps reducing the possibility of contamination, increasing the assay sensitivity, and improving the turnaround time [10], commercially available real-time PCR assays are just as expensive as the more standard nucleic acid viral load tests and rely on expensive, often dedicated equipment, that can only be used for these assays [10, 11]. For this reason, inexpensive and technologically simpler assays are still needed in resource-limited countries, where the new technology and equipments are currently not available. We generated RNA standards with HIV-1 and internal control sequences, using in vitro transcription method and evaluated its performance in a qRT-PCR for determining HIV-1 viral load

Materials and Methods

Evaluation of the HIV-1 and IC Transcripts

Results and Discussion