1. Quantification of Reverse Transcriptase Activity by Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral Vectors
- Author
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Massimo Pizzato, Veronica Iannucci, Evelien Naessens, Tom Taghon, Alessia Landi, Bruno Verhasselt, Hanne Vanderstraeten, Anouk Van Nuffel, and Jolien Vermeire
- Subjects
lcsh:Medicine ,law.invention ,chemistry.chemical_compound ,law ,Medicine and Health Sciences ,Pathology ,ASSAY ,Organic Chemicals ,lcsh:Science ,Polymerase chain reaction ,Multidisciplinary ,Titrimetry ,HIV diagnosis and management ,HIV Reverse Transcriptase ,Titer ,Real-time polymerase chain reaction ,HUMAN-IMMUNODEFICIENCY-VIRUS ,Viral Enzymes ,Quinolines ,Medicine ,Infectious diseases ,Viral Vectors ,PORCINE ENDOGENOUS RETROVIRUSES ,Research Article ,Clinical Pathology ,POLYMERASE-CHAIN-REACTION ,Genetic Vectors ,Viral diseases ,Biology ,Diamines ,Real-Time Polymerase Chain Reaction ,MONITOR RETROVIRUS ,Microbiology ,Sensitivity and Specificity ,REPLICATION-COMPETENT ,Virus ,Vector Biology ,Viral vector ,Cell Line ,Diagnostic Medicine ,VACCINES ,Complementary DNA ,Virology ,Viruslike Particles ,Humans ,INFECTIVITY ,Benzothiazoles ,lcsh:R ,HIV ,MAB CELL-CULTURE ,Molecular biology ,Reverse transcriptase ,Clinical Microbiology ,chemistry ,T-CELLS ,SYBR Green I ,HIV-1 ,lcsh:Q - 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.
- Published
- 2012