On-line version ISSN 1678-4596
SOUZA, Karina Koerich de et al. Optimization of PCR technique for detection of Actinobacillus pleuropneumoniae. Cienc. Rural [online]. 2008, vol.38, n.8, pp.2239-2244. ISSN 1678-4596. http://dx.doi.org/10.1590/S0103-84782008000800023.
The use of molecular methods based on PCR is important in Actinobacillus pleuropneumoniae detection, being able to identify the infection before the establishment of the disease in the herd. These methods have larger sensitivity when compared with traditional methods of bacteriological isolation, but they can suffer influence of substances that reduce the specificity of the test and resulting in inespecific amplifications. In order to reduce inespecific amplifications, observed when applied the PCR technique for the gene cpx in tonsil's tissue samples, the optimization was performed, in which different annealing temperatures were analyzed and introduced, in the technique, an antibody that binds to the enzyme Taq DNA Polimerase, increasing its specificity. In parallel, an experiment was performed in order to verify the inhibiting effect of the tonsil's tissue on the PCR results. For that, portions of tonsil from animals negative to the A. pleuropneumoniae were artificially contaminated with the reference sample of the sorotype 5B. The addition antibody for the enzyme Taq DNA Polimerase and the increase of the primers anneling temperature to 57°C reduced the inespecific amplifications. The results obtained in the experiment demonstrated a possible inhibiting effect of the tonsil's tissue in the PCR amplifications. Besides, amplifications depend on at least 675 UFC present in the aliquot of samples that will be used in PCR (equivalent to 1.35 x 105 UFC mL-1), therefore, samples tissue's fragments in initial infections and/or with few cells can result in false-negative.
Keywords : PCR; gene cpx; tonsilar tissue; Actinobacillus pleuropneumoniae; porcine pleuropneumonia.