title: Product-enhanced reverse transcriptase assay for replication-competent retrovirus and lentivirus detection.
abstract: The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24gag ELISA/gag PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Sastry
authors.forename: Yi
authors.initials: Y
authors.lastname: Xu
authors.forename: Lisa
authors.initials: L
authors.lastname: Duffy
authors.forename: Sue
authors.initials: S
authors.lastname: Koop
authors.forename: Aparna
authors.initials: A
authors.lastname: Jasti
authors.forename: Holger
authors.initials: H
authors.lastname: Roehl
authors.forename: Doug
authors.initials: D
authors.lastname: Jolly
authors.forename: Kenneth
authors.initials: K
authors.lastname: Cornetta
mesh_terms: D001681:Biological Assay / Q000379:methods; D016086:Lentivirus / Q000737:chemistry / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000032:analysis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014779:Virus Replication / Q000235:genetics
chemical_list: D012194:RNA-Directed DNA Polymerase