Detection and tentative identification of dominant mycoplasma species in cell cultures by restriction analysis of the 16S-23S rRNA intergenic spacer regions

Abstract

Mycoplasma (class Moliicutes) is one of the most common contaminants of cell cultures (McGarrity et aL, 1992). It is not always easy to find mycoplasma contamination in cell culturcs because the changes are not apparent. Viral s~ocks harvested from mycoplasma-contaminated celle cultures may be contaminated with a corresponding mycoplasma species; one recent case is that of the human immunodeficiency virus (HIV) type l which was grown in a lymphoblastoid cell line contaminated with Mycoplasma fermentans (Lemaitre et al., 1992). Live virus vaccines prepared from mycoplasmacontaminated cell cultures may become contaminated with mycoplasmas. Biopharmaceutical drugs produced usag animal cell cultures may also contain myceplasma cells. Mycoplasma contamination of virus vaccines and drugs may be a serious concern because mycoplasmas are currently suspected cofactors of AIDS (Lo, 1992). Another problem is that mycoplasmas can induce various cytokines including tumour necrosis factor, which may enhance HIV replication (Chowdhuri et aL, 1990). Most cytokineproducing cell lines are not only contaminated with mycoplasmas but also induced by mycoplasmas themselves (McGarrity et al., 1992). Thus it is imperative to examine mycoplasma contamination of cell cultures using reliable methods. Although various procedures are now available to detect mycoplasmas in cell cultures (Uphoff et al., 1992), serological and culture methods are still indispensable for the identification of mollicute species. Five mollicute species, M. fermentans, M. hyorhinis, M. orale, M. arginini, and Acholeplasma laidlawii, are known to account for more than 95 % of the mycoplasma contamination in cell cultures (McGarrity et aL, 1992). We developed a sent, tive and practical method to identify as v.c2! ,~ to detect these mycoplasma species which c o n taminate animal cell cultures.