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. 1999 May;29(3):221–227. doi: 10.1023/A:1008000210125

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

Haruhiko Kuno 1, Hiromi Ikeda 2, Masao Takeuchi 1, Touho Yoshida 1,✉
PMCID: PMC3463392  PMID: 19003345

Abstract

Reverse transcriptase (RT) is a good diagnostic tool for the detection of retroviruses. We have developed a simple and rapid assay for RT activity in culture supernatants. A 370-base RNA sequence from the tetracycline-resistance 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 chain reaction. A sensitivity test using purified recombinant RT of human immunodeficiency virus type 1 demonstrated that the detection limit of this method was 10-7–10-8 units of RT activity in 20 μl of a test sample (2 × 10-9–2 × 10-10 units ml-1). This method detected RT activity in unconcentrated supernatants of cell cultures infected with human T-cell leukemia virus, 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.

Keywords: cell culture, polymerase chain reaction, retrovirus, reverse transcriptase

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