L'uso dell'analizzatore genetico Applied Biosystems 3500 al fine di supportare il processo di caratterizzazione molecolare dei QUBs in ceppi di M. bovis nell'Istituto Zooprofilattico di Torino

The bovine tuberculosis (BT) is a zoonoses caused by the microorganism M. bovis. This disease is relevant on the Italian territory for what concerns cattle breeding and their slaughter for the meat industry. The pathology involves the chronic formation of tubercles in the respiratory tissue, affecting its function with cavitations rising. For the prevention of the transmission to humans and counteracting the damages to bovine zootechny, in 1995 the National Plan of eradication of bovine tuberculosis (D.L. 592, 15/12/95) established the procedure to control animals. The tuberculin test and γ-interferon measurement are the in vivo examinations provided for, in the regulation. The molecular procedure involves traditional techniques for the genomic characterization, such as PCR and gel electrophoresis. In particular, the first objective is the identification of isolated strains as belonging to genus Mycobacterium, to species M. avium or to M. tuberculosis or to the group of M. tb complex, in which M. bovis is included. The samples previously identified as members of M. tb complex are characterized in their genetic diversity through the analysis of specific genomic motifs, present in variable number of tandem repetitions (VNTRs). This thesis has the aim of setting the fragment analysis method to improve the molecular methodology, in particular on 7 QUBs (Queen's University Belfast VNTRs) loci. The use of Applied Biosystems 3500 Genetic Analyzer makes the results with more precision and objectivity, via Fragment Analysis Program. The experiments involved 8 ¿control¿ strains, used for the validation of the method and characterized for all the targets; and 20 experimental samples, involved in the concrete applications, such as in case of doubt or infrequent outcomes. In the validation phase, the end point results (gel reading) emerged equal to the sequencer data; this proved the efficacy of the method. For the applicability of it, the doubt results were resolved with a single value outcome, and the uncommon ones were confirmed. The instrument efforts the capillary electrophoresis and the GeneMapper Software displays the injection result with a plot (electropherogram) in which the major peak corresponds to the number of repetitions of the specific QUB motif. So, it is easier to understand and surer than traditional analyses, made by human operators. This method can overcome the difficulties of gel reading and the subjectivity of some interpretations.