Optimização da técnica de PCR para o diagnóstico da doença de Newcastle

Abstract

The Newcastle disease virus, known in the southern region of the country as “Mazungu”, attacks avian species. This virus has a genome that is made up of a single strand of RNA. Total RNA was extracted from 5 virus-infected positive amion fluid field samples by the hemagglutination inhibition test and 3 vaccine samples both types of samples by the TRIZOL Method. For its molecular diagnosis by the Polymerase Chain Reaction (PCR) technique, it was necessary to synthesize the complementary DNA molecule using the reverse transcription enzyme Superscript TMIII. The complementary DNA formed was immediately used as DNA for the first amplification (PCR I), followed by the second amplification (PCR II), in order to guarantee a more specific product, whose DNA corresponds to the product of PCR I. The PCR II samples were run on a 1% agarose gel, in which, after visualizing the bands of all samples, those that showed product amplification on the agarose gel were selected for use in PCR optimization by reducing concentrations of the enzyme Taq polymerase and MgCh- The results obtained indicate that PCR II without handling the reagents (standard PCR II) showed better results in terms of band visualization in all samples except one field sample. Vaccine samples tested positive across all ranges of concentrations of the intended reagents (TRADUÇÃO NOSSA)