Uso da técnica de southern blot/hibridização associada à reação em cadeia da polimerase para aumentar a sensibilidade no diagnóstico das infecções por hemoplasmas em gatos domésticos

Abstract

The aim of this study was to determine whether Southern Blot/Hybridization (SB) associated to Polymerase Chain Reaction (PCR) improves the sensitivity in the detection of hemoplasma DNA in domestic cats (Felis catus). Whole blood was collected in tubes containing the anticoagulant ethylenediamine tetra-acetic acid and DNA extracted from 149 animals. PCR was performed using species specific primers to amplify the 16S ribosomal RNA subunit of Mycoplasma haemofelis and 'Candidatus M. haemominutum' from these samples. Hybridization was performed using a 16S rDNA probes chemically labeled and the results were visualized using a chemiluminescent substrate addition followed by autoradiography. Eighteen (12.1%) of the 149 tested samples had a positive PCR result for hemoplasma species DNA. SB/hybridization technique showed that 24/149 (16.1%) samples were positive for hemoplasmas, confirming the 18 PCR-positive results and reveling six additional positive animals (p < 0.001). SB/hybridization method with specific probes was more sensitive than PCR performed alone, being complimentary to this technique to diagnose infections caused by feline hemoplasmas.