Standardisation of restriction fragment length polymorphism analysis for Mycobacterium avium subspecies paratuberculosis

DNA from 1008 strains of Mycobacterium avium subspecies paratuberculosis, d igested by restriction endonucleases PstI and BstEII, was hybridised with a standard IS900 probe prepared by PCR and labelled non-radioactively by ECL . DNA fingerprints were scanned by CCD camera and analysed using the softwa re Gel Compar (Applied Maths, Kortrijk, Belgium). Thirteen restriction frag ment length polymorphism (RFLP) (PstI) types were detected, which where des ignated as A, B, C, D, E, F, G, H, I, J, K, L and M in accordance with the study of Pavlik et al. (1995) [Pavlik, I., Bejckova, L., Pavlas, M., Rozsyp alova, V., Koskova, S., 1995. Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and hu man dependent strains of Mycobacterium paratuberculosis isolated in various localities. Vet. Microbiol. 45, 311-318]. Twenty RFLP (BstEII) types were detected and designated as C1-3, C5, C7-20, S1 and Il in accordance with th e study by Collins et al. 1990 [Collins, D.M., Gabric, D.M., de Lisle, G.W. , 1990. Identification of two groups of il Mycobacterium paratuberculosis s trains by restriction endonuclease analysis and DNA hybridization. J. Clin. Microbiol. 28, 1591-1596]. A combination of both RFLP (PstI) and RFLP (Bst EII) results revealed a total of 28 different RFLP types. All the RFLP type s and detailed protocols are available at Internet web site WWW...: http:/ /www.vri.cz/wwwrflptext.htm. (C) 1999 Elsevier Science B.V. All rights rese rved.