16S rRNA identification of camel Mycoplasma
Positive isolates were further confirmed for mycoplasmas by PCR
amplification of the 16S rRNA gene using Mycoplasma specific
primers (Table 1). PCR reaction was performed in a 50 μl volume for each
isolate, consisting of 5 µl of 50 ng of Mycoplasma DNA, 10 µl of
10 x Taq buffer (10 mM tris- HCl [pH 8.8], 50 mM KCl), 1 µl of 50 pM
of forward and reverse primers, 1.5 mM MgCl2, 1 µl of 2U
of Taq polymerase, 1 µl of 50 uM of each dNTP, and 31µl of DNase- RNase-
free, deionized water. The PCR reaction was performed in a thermal
cycler (Biometra TRIO, Jena, Germany) with an intial denaturation at
94oC for 5 min., followed by 35 cycles of denaturation
at 94oC for 1 min, annealing at 55oC
for 1 min., and extension at 72oC for 1.5 min with a
final extention at 72oC for 10 min.