Although the IS900 gene is considered specific for Mycobacterium avium subsp. paratuberculosis (MAP), a PCR directed against it still has to be confirmed with other methods, as cross-reactions with related IS elements in other mycobacteria have been demonstrated. Real-time PCR has the advantage over nested PCR in minimising the risk of contamination, but is still very sensitive. However, confirmation of positive samples by sequencing of amplicons cannot easily be achieved when using real-time PCR.We therefore developed two new real-time PCR systems targeting other parts of IS900. The new primers and probes were designed to be as specific as possible and to complement our primary PCR system. Special attention was paid to the similarity between IS900 and the equivalent gene in strain 2333. Another system of primers and probe was designed for the gene f57, also regarded specific for MAP. However, as the MAP genome contains multiple copies of IS900 but merely one copy of f57, this system ought to be less sensitive. The newly designed systems were tested on several mycobacterial strains to elucidate specificity. They are also being applied to clinical samples that are positive for MAP in our primary PCR system. Evaluation of the sensitivity is in progress.When applied on 77 non-MAP mycobacterial strains, one each of the alternative IS900 systems gave weak positive reactions to an isolate of M. avium subsp. avium from a horse and to an M. kubicae-like isolate from a cat, respectively. Strain 2333, that is positive with our primary system, showed negative with all the three new systems.The level of unspecific reactions in all of the four PCR systems is probably very low and the new systems should therefore be reliable for confirmation of the primary system.

Sponsorship

Attendance to this Congress was sponsored by the EU-funded project SSPE-CT-2004-501903