ISSN:
1573-0778
Keywords:
cell culture
;
polymerase chain reaction
;
retrovirus
;
reverse transcriptase
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
,
Process Engineering, Biotechnology, Nutrition Technology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1008000210125
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