Development of a species-specific DNA probe for Mycoplasma capricolum

A specific DNA probe for the detection and identification of Mycoplasma capricolum, one of the causative agents of contagious agalactia syndrome, was selected from a genomic library. It consists of a 900bp RsaI genomic fragment of M. capricolum (reference strain), cloned into the EcoRV site of the plasmid Bluescript. By using the appropriate stringency this radiolabelled probe reacts specifically with M. capricolum when tested by dot blot hybridization against various mycoplasmal DNAs. The current level of sensitivity of the 32P-labelled 900bp RsaI probe is 500 pg of homologous DNA, corresponding to 5 x 10(4) mycoplasmas. A non radioactive labelling method, using the digoxigenin-11-dUTP, was also tested. The specificity of the digoxigenin-labelled probe was equivalent to that obtained with the radioactive probe. However the sensitivity of detection decreased to 1 ng of homologous DNA detected, corresponding to 1 x 10(5) mycoplasmas. Tests performed with milk samples have demonstrated that the radioactive 900 bp RsaI probe indeed detected M. capricolum contained in milk. A positive signal was obtained when 10(5) M. capricolum were present in the spot.