PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium based on polymorphisms in IS1311

Point mutations in the IS1311 sequences from sheep and cattle strains of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) and M. avium subsp. avium (M. avium) were targeted to develop a polymerase chain reaction (PCR) that would be useful in the diagnosis and control of Johne's disease. Candidate PCR tests were evaluated for sensitivity, specificity and ease of interpretation of the restriction endonuclease analysis (REA) products. One IS1311 PCR, amplifying a 608 base pair product, was shown to be suitable when the amplified product was digested with Hinfl and Msel. The PCR detected 50 fg of template DNA from M. paratuberculosis strain 316 V, the equivalent of 10 organisms. The test was evaluated further using purified DNA from M. paratuberculosis and M. avium isolates and diagnostic samples including primary radiometric cultures. All 89 M. paratuberculosis samples were correctly identified and typed according to host species or IS900 restriction fragment length polymorphism (RFLP) type. All 28 isolates of M. avium were also correctly identified. A second PCR/REA strategy based on a shorter fragment of IS1311 was developed for formalin-fixed paraffin-embedded tissue samples. It correctly differentiated sheep and cattle strains of M. paratuberculosis in 27 tissue Sample's in which acid fast bacilli had been observed in Ziehl Neelsen stains and in which sufficient amplified product was present for REA with Hinfl. Both tests were specific for M. paratuberculosis when tested against 24 other mycobacterial species. These simple and rapid tests can be used on a range of diagnostic samples for the confirmation of Johne's disease and will be of benefit in control and eradication programmes for this disease. (C) 1999 Academic Press.