Detection and identification of Mycoplasma conjunctivae in infectious keratoconjunctivitis by PCR based on the 16S rRNA gene

A specific PCR assay based on unique sequences of the rrs genes (16S rRNA) of Mycoplasma conjunctivae was developed For direct detection and identific ation of this pathogen from clinical material. DNA from eve swabs was ampli fied after a simple lysis step Lv either a single PCR with the M. conjuncti vae specific primer pair McoR1 and McoF1, or Lv a nested PCR with the Mycop lasma genus specific primer pair MOLIGEN1-L and 16UNI-R in the first step a nd McoR1 and McoF1 in the second step. The specificity of the primer pair M coR1 and hIcoF1 was verified with purified DNA from the type strain, from 1 7 field isolates of M. conjunctivae and from several Mollicutes which are p hylogenetically related to ill. conjunctivae Or which can be isolated from the same host animals. This method identified mycoplasma isolates from goat , sheep, ibex and chamois originating from different countries as M. conjun ctivae. No cross amplifications with other mycoplasmas which are related to M. conjunctivae were observed. Eve swab samples containing known numbers of M. conjunctivae cells were ana lysed after direct lysis of the material. The detection level was estimated to be 20 cells per swab when the nested PCR procedure was used and 2 x 10( 5) by the single PCR method. In an experimental infection model of sheep, t he nested PCR method for detection of M. conjunctivae gave results which we re comparable to mycoplasmal culture. These are the implications for diagno stic purposes: M conjunctivae isolates can be identified by the one-step PC R method, whereas for detection and identification of M. conjunctivae in cl inical material the two-step method should be used (higher sensitivity).