Different strategies for molecular differentiation of Mycobacterium bovis strains isolated in Sardinia, Italy

Abstract

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.

FIG. 1

Southern blot of chromosomal DNAs of M. bovis strains showing the representative patterns observed after hybridization with the 0.45-kb BamHI-SalI fragment of IS6110 used as a probe and HindIII-digested λ DNA. Lanes: M, HindIII-digested λ DNA; 1, strain 3; 2, strain 30; 3, strain 92; 4, strain 94; 5, strain 95; 6, strain 0.

FIG. 2

(a) Agarose gel electrophoresis (1.8% Methaphor agarose) of HaeIII restriction digests of the amplification products of M. bovis isolates obtained with the PCR ribotyping method. Lanes: 1, strain 0; 2, strain 30; 3, strain 1; 4, strain 2; 5, strain 3; 6, strain 4; 7, strain 6; 8, strain 7; 9, strain 10; 10, strain 11; 11, strain 13; 12, strain 14; 13, strain 15; 14, strain 16; 15, strain 17; 16, strain 91; 17, strain 92; 18, strain 94; 19, strain 95; 20, strain 97; 21, strain 98; 22, strain 114; 23, strain S1; 24, ATCC 27290; M, 100-bp molecular weight marker ladder (Amersham, Life Technologies). (b) Agarose gel electrophoresis (1.8% Methaphor agarose) of PvuII restriction digests of the amplification products of M. bovis isolates obtained by PCR ribotyping. Strains are as in panel a. (c) Agarose gel electrophoresis (1.8% Methaphor agarose) of DNA amplification of M. bovis isolates obtained by the PCR method using ERIC primers. Strains are as in panel a. (d) Agarose gel electrophoresis (1.8% Metaphore agarose) of DNA amplification of clinical isolates obtained by the PCR method using the (GTG)₅ oligonucleotide. Strains are as in panel a.