[Cloning universal probes for detecting mycoplasma contamination of cell cultures]

To obtain the universal polynucleotide hybridization probes for testing mycoplasmal contaminations in cell cultures, we have cloned several DNA fragments from the srRNA gene of Acholeplasma laidlawii. Before cloning, in order to exclude cross-hybridization of these probes with eukaryotic rRNA, the thermodynamic parameters of duplex formation between DNA complementary to mycoplasmal rRNA and eukaryotic rRNA had been studied. Using a set of computer methods, the region which forms weak heteroduplexes with eukaryotic srRNA was revealed. This region occupies positions 250 to 550 position of the mycoplasmal srRNA. Three different DNA fragments which include the region were generated in PCR, cloned in pUC18, and their hybridization characteristics were evaluated. In appropriate hybridization conditions the probes hybridize with all mycoplasmal RNAs studied without cross-hybridization with eukaryotic ribosomal RNA and DNA, and allow one to detect virtually any mycoplasmas (or any prokaryote) in cell cultures. Blot-hybridization of universal probes with mycoplasmal DNA digested by BsuRI allows one to identify the different species of mycoplasmas.