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1.6 BIOLOGICAL SCIENCES

Abstract

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping and more recently, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing. Strain typing of Mycobacterium bovis isolates based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) and on spoligotyping was evaluated in this study and these typing methods were compared with restriction fragment length polymorphism (RFLP) typing. A total of 386 M. bovis isolates from cattle, badgers and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS) and direct repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (2163a, 2163b, 2165, 4052, 2996 and 1895) was performed on the samples. RFLP was the method that gave the greatest differentiation of strains with a Hunter Gaston discriminatory index (HGDI) of 0.927, the HGDI recorded for MIRU-VNTR was marginally lower at 0.918 and spoligotyping was the least discriminatory method with a HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140 there was a much lower level of concordance between RFLP and MIRU-VNTR typing compared to groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol the number of VNTR loci could be reduced to four (2163a, 2163b 2165 and 4052) while maintaining a high level of strain differentiation.

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