Rapid identification of all known retroviral reverse transcriptase sequences with a novel versatile detection assay

We have developed a highly sensitive, universal assay that allows detection as well as identification of all known retroviral reverse transcriptase (R T)-related nucleic acids in a biological sample by a single two-step experi ment. The assay combines polymerase chain reaction (PCR) and reverse dot-bl ot hybridization (RDBH), using an array of immobilized synthetic retrovirus -specific oligonucleotides and two sets of mixed oligo primers (MOPs), Thes e primers were derived from highly conserved motifs found in all known reve rse transcriptase genes. The PCR/RDBH assay was used for qualitative analys es of human endogenous retrovirus (HERV) transcription in peripheral blood mononuclear cells (PBMCs) and in particles released by the human mammary ca rcinoma-derived cell line T47D, Sensitivity was further demonstrated by det ection of down to 10 copies of pig endogenous retrovirus (PERV) DNA in huma n cDNA samples. Therefore, this assay is particularly useful for the identi fication of retroviral sequences in xenografts as well as in recipients of xenografted tissues and organs. Moreover, it is a valuable tool to detect r etroviral transcripts and particles in cell cultures used for production of therapeutic polypeptides, The assay is further suitable for monitoring vec tor preparation used in human gene therapy to exclude transfer of copackage d endogenous retroviruses into target cells.