Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors

Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dyn...

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Main Authors: Vermeire, Jolien, Naessens, Evelien, Vanderstraeten, Hanne, Landi, Alessia, Iannucci, Veronica, Van Nuffel, Anouk, Taghon, Tom, Pizzato, Massimo, Verhasselt, Bruno
Format: Online
Language:English
Published: Public Library of Science 2012
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515444/
id pubmed-3515444
recordtype oai_dc
spelling pubmed-35154442012-12-07 Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors Vermeire, Jolien Naessens, Evelien Vanderstraeten, Hanne Landi, Alessia Iannucci, Veronica Van Nuffel, Anouk Taghon, Tom Pizzato, Massimo Verhasselt, Bruno Research Article Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories. Public Library of Science 2012-12-05 /pmc/articles/PMC3515444/ /pubmed/23227216 http://dx.doi.org/10.1371/journal.pone.0050859 Text en © 2012 Vermeire et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Vermeire, Jolien
Naessens, Evelien
Vanderstraeten, Hanne
Landi, Alessia
Iannucci, Veronica
Van Nuffel, Anouk
Taghon, Tom
Pizzato, Massimo
Verhasselt, Bruno
spellingShingle Vermeire, Jolien
Naessens, Evelien
Vanderstraeten, Hanne
Landi, Alessia
Iannucci, Veronica
Van Nuffel, Anouk
Taghon, Tom
Pizzato, Massimo
Verhasselt, Bruno
Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
author_facet Vermeire, Jolien
Naessens, Evelien
Vanderstraeten, Hanne
Landi, Alessia
Iannucci, Veronica
Van Nuffel, Anouk
Taghon, Tom
Pizzato, Massimo
Verhasselt, Bruno
author_sort Vermeire, Jolien
title Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
title_short Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
title_full Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
title_fullStr Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
title_full_unstemmed Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
title_sort quantification of reverse transcriptase activity by real-time pcr as a fast and accurate method for titration of hiv, lenti- and retroviral vectors
description Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.
publisher Public Library of Science
publishDate 2012
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3515444/
_version_ 1611938408185200640

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