title: Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors.
abstract: 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.
authors.affiliation: Department of Clinical Chemistry, Microbiology, and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Jolien
authors.initials: J
authors.lastname: Vermeire
authors.forename: Evelien
authors.initials: E
authors.lastname: Naessens
authors.forename: Hanne
authors.initials: H
authors.lastname: Vanderstraeten
authors.forename: Alessia
authors.initials: A
authors.lastname: Landi
authors.forename: Veronica
authors.initials: V
authors.lastname: Iannucci
authors.forename: Anouk
authors.initials: A
authors.lastname: Van Nuffel
authors.forename: Tom
authors.initials: T
authors.lastname: Taghon
authors.forename: Massimo
authors.initials: M
authors.lastname: Pizzato
authors.forename: Bruno
authors.initials: B
authors.lastname: Verhasselt
mesh_terms: D052160:Benzothiazoles; D002460:Cell Line; D003959:Diamines; D005822:Genetic Vectors / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017186:Titrimetry / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase