Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900 , IS901, IS1245 and IS901 -flanking region PCR with internal standards Milan Bartos a, * , Pavel Hlozek a , Petra Svastova a , Lenka Dvorska a , Tim Bull b , Ludmila Matlova a , Ilona Parmova a,c , Isolde Kuhn a , Janine Stubbs a , Monika Moravkova a , Jaromir Kintr a , Vladimir Beran a , Ivan Melicharek d , Matjaz Ocepek e , Ivo Pavlik a a Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic b St. George’s Hospital, Medical School, Cranmer Terrace, London, United Kingdom c State Veterinary Diagnostic Institute, Sidlistni 24, 165 03 Prague-Lysolaje, Czech Republic d State Veterinary Diagnostic Institute, Akademicka 3, 949 01 Nitra, Slovakia e Veterinary Faculty, University of Ljubljana, Gerbiceva 60, 61 000 Ljulbljana, Slovenia Received 5 January 2005; received in revised form 11 May 2005; accepted 24 May 2005 Available online 2 August 2005 Abstract From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n = 961), Mycobacterium a. avium (n = 677), Mycobacterium a. silvaticum (n = 5), and Mycobacterium a. hominissuis (n = 1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n = 2), Mycobacterium bovis (n = 13), M. bovis BCG (n = 4), and Mycobacterium caprae (n = 10) were examined. From other mycobacterial species Mycobacterium intracellulare (n = 60) and atypical mycobacteria (n = 256) including Mycobacterium fortuitum, Mycobacterium chelonae , Mycobacterium scroful- aceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10 1 , 10 3 , 10 2 , and 10 2 molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.05.009 * Corresponding author. Tel.: +420 5 3333 1615; fax: +420 5 4121 1229. E-mail address: bartos@vri.cz (M. Bartos). URL: http://www.vri.cz (M. Bartos). Journal of Microbiological Methods 64 (2006) 333 – 345 www.elsevier.com/locate/jmicmeth