DNA probes for detection and identification of mycoplasmas (Mollicutes)

Abstract

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.