Isolation of a specific DNA fragment and development of a PCR-based methodfor the detection of Mycobacterium genavense

The rise of Mycobacterium genavense infections is making identification eve r mere important for diagnosis and treatment. Moreover, isolation and ident ification of M. genavense are made difficult by the lack of growth on solid media and by its low generation rate in BACTEC liquid media. Thus, amplifi cation by PCR or similar techniques represents the only possibility of dete cting and identifying M. genavense from tissue samples. In order to set up a simple and species-specific method based on the use of PCR and non-radioa ctive hybridization technique, we decided to search for and clone a specifi c DNA fragment of this bacterial species. In the present study, a 1734-bp f ragment was isolated. This fragment was found to be highly specific for M. genavense strains. A species-specific pair of primers (MG22 and MG23) and t wo oligonucleotide probes (MG18 and MG19) were selected. They were successf ully used to amplify and detect a 155-bp DNA fragment from the 13 available strains of M. genavense which were isolated from clinical specimens or fro m birds. Conversely, the primers and probes did not hybridize with DNA from any of the 20 other mycobacterial species tested. It is worth noting that the chosen primers and probes did not hybridize with DNA of M. simiae, alth ough it is closely related to M. genavense. The present PCR technique uses species-specific primers for M. genavense. Followed by a non-radioactive hy bridization technique on microplates it is able to distinguish M. genavense from other mycobacteria in one step, without sequencing or restriction ana lysis. On the basis of the Southern blot hybridization, PCR and sandwich hy bridization results, we concluded that the isolated 1.7-kb sequence was spe cific for the M. genavense chromosome. The method developed here for M. gen avense identification uses a simple methodology and commonly available reag ents. Furthermore it can be easily automated. (C) 1999 Published by Elsevie r Science B.V. All rights reserved.