A REVERSE TRANSCRIPTASE-POLYMERASE CHAIN-REACTION ASSAY FOR THE DETECTION AND QUANTITATION OF MURINE RETROVIRUSES

Specific hybridization primers for the PCR assay were developed to det ect the presence of the ecotropic, xenotropic, and mink cell focus-for ming classes of murine leukemia viruses (MuLVs) in samples derived fro m cultured cells and cell-free supernatants. The primers, which were t ested against reference viruses from all three classes and two subclas ses and accurately identified each class present, were used to charact erize the endogenous expression of MuLV-related sequences in a number of murine and mink cell lines. Two murine/murine hybridomas were shown to contain expressed retroviral sequences from all three classes. The murine cell lines SC-1, Balb/c 3T3, and NIH 3T3, were found to consti tutively express sequences from many of the MuLV classes. These MuLV-r elated sequences were not expressed in the Mus dunni or mink lung cell lines. When these primers were used in a quantitative PCR assay to de termine the retroviral content of hybridoma supernatants, the values w ere less variable than those obtained by transmission electron microsc opy (TEM). This assay can be adapted to detect and quantitate any vira l contaminant in cell culture supernatants, ascites fluids, process va lidation samples, and final products.