Aplicação do PCR para a detenção de Trypanossoma Congolense em T. Vivas no diagnóstico molecular da Tripanosomose bovina

Abstract

Trypanosomiasis is one of the biggest constraints on agricultural activities based on livestock in Africa. Mozambique is one of 37 African countries affected by the disease. The country is part of a vast territory inhabited by tsetse flies (Glossina spp) vectors of the disease - the tsetse belt. Glossina spp cyclically transmit protozoa of the genus Trypanosoma, which are the pathogenic hemoparasites that cause disease. In Africa there are 11 species, and in Mozambique, as agents of bovine trypanosomosis, the species Tryoanosoma congolense and T. vivax occur mainly. For the diagnosis of trypanosomosis, several techniques are applied that differ in sensitivity, ease and cost of application, factors that together with the purpose of diagnosis lead the choice of the appropriate technique. In this context, the techniques for detecting trypanosomes based on DNA (Nucleic Acid Hybridization and PCR) appear as techniques of high sensitivity and specificity compared to all others (parasitological, live culture, antigen detection, serological), being recommended for epidemiological and typing/distribution studies. In the present work, the applicability of PCR in the laboratory diagnosis of bovine trypanosomiasis caused by T. congolense and T vivax was evaluated, concluding that the application of this technique in the country should be encouraged, although not routinely due to the high costs, but for specific studies. The performance of universal diagnostic primers (ITS 1 cf/br) and species-specific diagnostic primers (PCR Cat L primers) was evaluated, having concluded that the latter are better in terms of sensitivity, quality and quantity. of the amplicons. The influence of the DNA extraction method on PCR performance was also evaluated in relation to two methods - extraction of whole blood and extraction by boiling of impregnated palel filter discs - having concluded that the latter is completely inadvisable as it has very low levels of sensitivity regardless of the type of primer, although this is more noticeable with universal primers. The analytical sensitivity of the PCRs was evaluated through serial dilutions of the DNA, however, it did not obtain conclusive results for the PCR ITS 1, while for the PCR Cat L this was done in the 10x dilution of the DNA extracted from impregnated discs(TRADUÇÃO NOSSA)