Evaluation of tRNA gene PCR for identification of mollicutes.
We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.
Stakenborg
Tim
T
Veterinary and Agrochemical Research Center, Brussels, Belgium.
Vicca
Jo
J
Verhelst
Rita
R
Butaye
Patrick
P
Maes
Dominiek
D
Naessens
Anne
A
Claeys
Geert
G
De Ganck
Catharine
C
Haesebrouck
Freddy
F
Vaneechoutte
Mario
M
D000127:Acholeplasma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D000829:Animals, Domestic / Q000382:microbiology; D015373:Bacterial Typing Techniques; D001717:Birds / Q000382:microbiology; D002417:Cattle; D004269:DNA, Bacterial / Q000032:analysis; D020459:Genes, rRNA; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012343:RNA, Transfer / Q000235:genetics*; D008973:Tenericutes / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D014509:Ureaplasma / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012343:RNA, Transfer