Detection of Mycobacterium avium subsp paratuberculosis in tissue samples by single, fluorescent and nested PCR based on the IS900 gene

The aim of this study was to determine if fluorescent PCR could be used ins tead of nested PCR, for the detection of Mycobacterium avium subsp. paratub erculosis (M. paratuberculosis) in clinical specimens, to improve the sensi tivity without increasing the risk for cross-contamination. We investigated and compared the sensitivity of single PCR, fluorescent PCR and nested PCR for the detection of IS900, an insertion sequence specific for M. paratube rculosis. A previously described extraction method for clinical specimens, based on xylene, was evaluated regarding its suitability for routine diagno stic work. The sensitivity of each PCR system was assessed by analysing a s erial dilution of M. paratuberculosis DNA. To improve the reliability of th e PCR and to facilitate the interpretation of the PCR results, a positive i nternal control molecule ("mimic") was developed and used for single and fl uorescent PCR. In nested PCR, an existing mimic was used. The efficiency of recovering DNA of M. paratuberculosis from clinical specimens by the extra ction method and detection of the organism by PCR was studied by analysing spiked ileum mucosa specimens. The final evaluation was performed on sevent een ileum mucosa specimens, previously found positive for M, paratuberculos is by bacterial culture. Twelve of the samples were positive by fluorescent PCR and nested PCR, and 10 samples were positive by single PCR. The use of mimics showed inhibition in specimens harbouring few M. paratuberculosis o rganisms, illustrating the effect of inhibitory substances in combination w ith small amounts of M. paratuberculosis DNA. We conclude that the extracti on method was not adequate to recover small amounts of M. paratuberculosis and that inhibitory substances were still present in the processed specimen s, but that the method is useful for identifying positive samples. Fluoresc ent PCR was a suitable alternative to both single PCR and nested PCR for th e detection of M. paratuberculosis. (C) 2001 Elsevier Science B.V. All righ ts reserved.