title: Comparison of TaqMan real-time PCR and p24 Elisa for quantification of in vitro HIV-1 replication.
abstract: In this study, TaqMan PCR was used to assess viral replication of HIV-1 infected cells in vitro. This PCR technique was compared with p24 ELISA as a standard method to monitor HIV-1 replication in cell culture. Hut78 T-lymphoblastoid cells were infected with different titres of HIV-1(IIIb) (MOI 0.05-0.0005). The course of HIV-1 replication was monitored by determination of p24 concentrations by ELISA in cell culture supernatants and by quantitation of HIV-1 gag RNA by TaqMan RT-PCR. Additionally, the number of HIV-1 proviral copies was assessed by TaqMan PCR. Monitoring of HIV-1 replication by p24 ELISA and TaqMan RT-PCR revealed comparable kinetics of infection. Both methods provided similar data on the exponential increase and on plateauing of HIV-1 replication. Furthermore, both methods were equally sensitive. However, a 7 log linearity of TaqMan HIV-1 gag PCR was demonstrated without dilution of the specimen, in contrast to p24 ELISA, where because of its narrow range of detectable p24 concentrations, sample dilution was necessary. Although determination of the number of proviral copies by TaqMan PCR does not measure HIV-1 replication, the kinetics of proviral copy number following in vitro inoculation of cells with HIV-1 was nearly the same as the kinetics of HIV-1 RNA copy numbers. In conclusion, TaqMan real-time RT-PCR was demonstrated as a reliable and sensitive tool to quantify and monitor HIV-1 replication in cell culture. It is suggested, therefore, that this technique be an alternative method to monitor HIV-1 replication in vitro.
authors.affiliation: Medizinische Klinik III der Johann Wolfgang Goethe Universität, Theodor-Stern Kai 7, D-60590, Frankfurt, Germany. sal.klien@t-online.de
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Klein
authors.forename: Susanne
authors.initials: S
authors.lastname: Karsten
authors.forename: Brigitte
authors.initials: B
authors.lastname: Rüster
authors.forename: Christian
authors.initials: C
authors.lastname: Klebba
authors.forename: Martine
authors.initials: M
authors.lastname: Pape
authors.forename: Oliver G
authors.initials: OG
authors.lastname: Ottmann
authors.forename: Dieter
authors.initials: D
authors.lastname: Hoelzer
authors.forename: Willi K
authors.initials: WK
authors.lastname: Roth
mesh_terms: D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015683:Gene Products, gag / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism; D014779:Virus Replication
chemical_list: D015683:Gene Products, gag; D016655:HIV Core Protein p24; D012367:RNA, Viral; D019914:Taq Polymerase