A simple and rapid reverse transcriptase assay for the detection of retroviruses in cell cultures

Reverse transcriptase (RT) is a good diagnostic tool for the detection of r etroviruses. We have developed a simple and rapid assay for RT activity in culture supernatants. A 370-base RNA sequence from the tetracycline-resista nce gene in pBR322 plasmid DNA was used as a template for RT-mediated cDNA synthesis. To detect the resultant cDNA, we used the nested polymerase chai n reaction. A sensitivity test using purified recombinant RT of human immun odeficiency virus type 1 demonstrated that the detection limit of this meth od was 10(7)-10(8) units of RT activity in 20 mu l of a test sample (2 x 10 (-9)-2 x 10(-10) units ml(-1)). This method detected RT activity in unconce ntrated supernatants of cell cultures infected with human T-cell leukemia v irus, Moloney murine leukemia virus, Moloney murine sarcoma virus, or Rous sarcoma virus. This nonisotopic method provides results within 10 h and is useful for quality control to detect retroviruses in cell cultures.