There is a test requirement (CFR Section 21:610.30) for the presence of mycoplasma in all mammalian cell lines used for the production of viral vaccines for human use and in """"""""Points to be considered"""""""" to test for mycoplasma in other human biologics produced in cell substrates and grown in vitro. We have developed a system suitable for studying the DNA of M. hyorhinis in situ, that is, without growing the mycoplasma axenically. Our system takes advantage of three facts: mycoplasmas coisolate with mitochondria; their DNA can be differentially labeled from nuclear DNA; the restriction pattern of mycoplasmal DNA can be easily differentiated from that of the mitochondrial DNA even in mixtures of the two DNAs. This technology should be useful for studying other strains of mycoplasma as well and could obviate the need for synthetic media to culture them. Additionally, this technology can provide an effective means to investigate mycoplasma-tissue cell interactions, particularly with the fastidious, pathogenic, noncultivable mycoplasma species and strains. Mycoplasma hyorhinis coisolates with the mitochondria of the cells in which it is carried as an infection. Since both mitochondria and mycoplasmas synthesize DNA by using the prokaryotic DNA polymerase gamma, the use of aphidicolin, which inhibits eukaryotic DNA polymerase alpha, allows for selective synthesis of mycoplasmal and mitochondrial DNA. The restriction patterns of mitochondria and mycoplasmas can easily be differentiated from each other in mixtures of both DNAs. Thus, it is possible to study the molecular biology of this noncultivable mycoplasma in situ rather than after growth in artificial media, with its potential genetic consequences during adjustment to axenic growth.
