DNA extraction
Isolates that only reacted with M. bovis antisera were selected. The bacterial lysates used as templates for the PCR were prepared as follows: A loopful of bacteria from a fresh overnight culture on a tryptic soy agar plate (Difco, Detroit, MI, USA) was re-suspended homogeneously in 200µl of sterile water, and the mixture was boiled at 1000C for 5min to release the DNA and centrifuged. The supernatant was used as a template for PCR mixture. Confirmation of these isolates as species of M. bovis was achieved using two PCR-based assays: 1) PCR products were generated from each isolate using primers that amplify a 16S rRNA sequence specific for Mycoplasmaspecies (Yleana et al., 1995); 2) then the Mbo gene was amplified using MboF/MboR primers which is a confirmation of M. bovis(Subramaniam et al., 1998) (Table 1).
The DNA of the M. bovis type strain ATCC 25523/ PG45 served as a positive control, while nuclease free water was used as negative control.