Real-time RT-PCR detection of retroviral contaminations of cells and cell lines.

We have developed a fast and sensitive on-line detection method for retroviruses using the PCR technology. The assay utilizes the endogenous reverse transcriptase activity in retroviral particles. In the presence of active reverse transcriptase, bacteriophage MS2 RNA is transcribed into cDNA and is subsequently amplified in a SYBR-Green-type LightCyclertrade mark reaction. The method allows a qualitative and quantitative monitoring of RT-activity, is several orders of magnitude more sensitive than a standard RT assay and has a time requirement of 2.5 hours from harvest to result. The methodis useful for monitoring of cells and cell-derived products, viral vectors and recombinant proteins for the presence ofreplication-competent retroviruses (RCRs).