Isolation of a Rhizobium galegae strain-specific DNA probe

We have isolated a strain-specific DNA probe from the strain Rhizobium galegae HAMBI 1174 by a subtraction hybridization procedure followed by PCR amplification and DNA cloning. The specificity of the 342-bp DNA probe (P3) was tested in dot blot or Southern blot hybridizations against total genomic DNA of 41 bacterial strains (21 of them belong to R. galegae, 15 to other Rhizobium species and five to other bacterial species). Only the samples from four R. galegae strains, which are different isolates but identical to the strain HAMBI 1174, hybridized with the probe. The P3 probe was sequenced and PCR primers were designed based on its sequence. PCR amplification from purified total genomic DNA of 52 strains and subsequent hybridization with the P3 probe proved that the primers are strain specific.