Evaluation of amplified rDNA restriction analysis (ARDRA) for the identification of Mycoplasma species
Stakenborg, T × Vicca, Jo Butaye, P Maes, Dominique De Baere, T Verhelst, R Peeters, J de Kruif, A Haesebrouck, F Vaneechoutte, M #
BMC Infectious Diseases vol:5 issue:46 pages:1-10
Background: Mycoplasmas are present worldwide in a large number of animal hosts. Due to their
small genome and parasitic lifestyle, Mycoplasma spp. require complex isolation media.
Nevertheless, already over 100 different species have been identified and characterized and their
number increases as more hosts are sampled. We studied the applicability of amplified rDNA
restriction analysis (ARDRA) for the identification of all 116 acknowledged Mycoplasma species and
Methods: Based upon available 16S rDNA sequences, we calculated and compared theoretical
ARDRA profiles. To check the validity of these theoretically calculated profiles, we performed
ARDRA on 60 strains of 27 different species and subspecies of the genus Mycoplasma.
Results: In silico digestion with the restriction endonuclease AluI (AG^CT) was found to be most
discriminative and generated from 3 to 13 fragments depending on the Mycoplasma species.
Although 73 Mycoplasma species could be differentiated using AluI, other species gave
undistinguishable patterns. For these, an additional restriction digestion, typically with BfaI
(C^TAG) or HpyF10VI (GCNNNNN^NNGC), was needed for a final identification. All in vitro
obtained restriction profiles were in accordance with the calculated fragments based on only one
16S rDNA sequence, except for two isolates of M. columbinum and two isolates of the M. mycoides
cluster, for which correct ARDRA profiles were only obtained if the sequences of both rrn operons
were taken into account.
Conclusion: Theoretically, restriction digestion of the amplified rDNA was found to enable
differentiation of all described Mycoplasma species and this could be confirmed by application of
ARDRA on a total of 27 species and subspecies.