A cloned DNA probe for the detection of Mycobacferium pamtuberculosis

Abstract

DNA extracted from Mycobacterium paratuberculosis, which had been isolated from a cow with clinical Johne's disease, was used to make a gene library in the Escherichia coli expression vector phage λgt11. Plaque-lifts were made from the library onto nitrocellulose membranes. These were screened by differential hyhridization using radiolabelled chromosomal DNA from M. paratuberculosis and Mycobacterium phlei. By this method six recomhinants that hybridized to M. paratuberculosis but not to M. phlei were identified. Three of these, designated λgt-R3, λgt-R4 and λgt-R5, containing DNA inserts of 2.5,1.5 and 3.7 kilobases (kb), respectively, were chosen for further analysis of their insert specificities. Following restriction with the endonucleases EcoRI and BamHI, the digestion fragments from the three recombinants were transferred to nitrocellulose membranes and probed with radiolabelled DNA from M. pamtuberculosis and M. phlei. As expected, M. paratuberculosis DNA hybridized to all the fragments. M. phlei DNA hybridized to both the fragments that were generated from λgt-R3, to the single fragment from λgt-R4 and to two of the three fragments generated from λgt-R5. The fragment with which M. phlei DNA failed to hyhridize was 0.45 kb in length. Multiple copies of this fragment were made in the plasmid pGEM-2; the plasmid DNA was then harvested and radiolabelled. Designated PAM-1, the radiolabelled material hybridized to a 3.7 kb fragment of EcoRI-digested M. paratuberculosis and to 2.2 kb fragments of similarly digested M. avium serovars 2 and 3. PAM-1 did not hybridize to DNA from the other four mycobacterial species examined or from Nocardia asteroides. The restriction fragment length polymorphism thus demonstrated distinguishes M. paratuberculosis from M. avium serovars 2 and 3.