Sensitive Detection of Mycoplasmas in Cell Cultures by Using Two-Step Polymerase Chain Reaction

Abstract

Mycoplasma contamination of cell cultures has been a serious problem in biomedical research. The incidence of mycoplasma contamination is higher than might be expected, and it is difficult to eliminate mycoplasmas from contaminated cell cultures (Barile et al., 1978; McGarrity and Kotani, 1985). Several techniques for the detection of mycoplasma contamination of cell cultures have been described (Barile and McGarrity, 1983). They are the direct culture procedure (Ogata and Koshimizu, 1967), enzyme activity tests (Barile and Schimke, 1963; Levine, 1972; Uitendall et al., 1979), fluorescent staining by DNA-binding dyes (Chen, 1977). The direct culture technique takes time and sometimes fails to detect fastidious mycoplasmas such as Mycoplasma hyorhinis (Hopps et al., 1973), and the enzyme activity test frequently leads to false results. Immunoenzyme techniques (Chasey and Woods, 1984; Kotani and McGarrity, 1985), and DNA-DNA hybridization by Southern blotting (Razin et al., 1984) or by using hydroxyapatite columns (Harasawa et al., 1986) have been reported as alternative and useful methods.