RAPID RETROVIRUS TITRATION USING COMPETITIVE POLYMERASE CHAIN-REACTION

A quantitative polymerase chain reaction (PCR) procedure has been deve loped for rapid retrovirus titration, This procedure, which is based o n the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantific ation of viral RNA genomes in retrovirus-producing cell clone supernat ants and of proviral DNA molecules formed at 24 h after infection of d ifferent reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in co mplete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the produ cer cell clone supernatants is a poor predictor or the actual efficien cy of infection. Repeated competitive PCR experiments for provirus cop y number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, s uggesting stable integration in to the host genome. The developed proc edure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the e fficiency of infection of any target cell type, thus overcoming the pr oblem of the dependence of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony for mation of a reference cell line.