Quantitative PCR-ELAHA for the determination of retroviral vector transduction efficiency

Current methods to detect transduction efficiency during the routine use of integrating retroviral vectors in gene therapy applications may require th e use of radioactivity and usually rely upon subjective determination of th e results. We have developed two competitive quantitative assays that use a n enzyme-linked, amplicon hybridization assay (ELAHA) to detect the product s of PCR-amplified regions of transgene from cells transduced with Moloney murine leukemia virus vectors. The quantitative assays (PCR-ELAHA) proved t o be simple, rapid, and sensitive, avoiding the need for Southern hybridiza tion, complex histochemical stains, or often subjective and time-consuming tissue culture and immunofluorescence assays. The PCR-ELAHA systems can rap idly detect proviral DNA from any retroviral vector carrying the common sel ective and marker genes neomycin phosphotransferase and green fluorescent p rotein, and the methods described are equally applicable to other sequences of interest, providing a cheaper alternative to the evolving real-time PCR methods. The results revealed the number of copies of retrovector provirus present per stably transduced cell using vectors containing either one or both qPCR targets.