Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes

Abstract

Five polymerase chain reaction (PCR) primer pairs were synthesized on the basis of the aligned 16S-like rRNA sequences of eukaryotes or 16S rRNA sequences of eubacteria, Mollicutes, and intracellular organelles. These PCR primer pairs had high sequence homology to the conserved 16S rRNA genes of various culturable and nonculturable Mollicutes, but less sequence homology to the eukaryotic nuclear 16S-like rRNA or 16S rRNA genes of intracellular organelles. Full-length 16S rRNA genes and partial-length 16S rRNA genes of evolutionarily variable regions were successfully amplified when DNA preparations from culturable Mollicutes such as Mycoplasma flocculare and three Spiroplasma strains and nonculturable Mollicutes associated with various plant diseases were used as PCR templates. Amplifications were not detected when Escherichia coli genomic DNA and DNA preparations from healthy plants were used under high stringency annealing conditions in thermocycling. The results suggest the possibility that 16S rRNA genes of culturable and nonculturable Mollicutes can be amplified for detection and for a phylogenetic study using crude Mollicutes DNA preparations under appropriately controlled thermocycling conditions.