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.