Desenvolvimento e otimização de métodos de Biologia Molecular para o diagnóstico de Leishimania chagasi e Helicobacter pylori

by Evans Osses, Ingrid Solange

Abstract (Summary)
In this work were developed and improved diagnostic methodologies for two pathogenic agents with high incident in Brazil and which diagnostic are based in invasive methodologies: Helicobacter pylori (H. pylori) and Leishmania chagasi (L. chagasi). The Helicobacter pylori is a gram (-) bacteria, main cause of peptic ulcer and malignant gastritis, which it diagnostic is does preferentially through endoscopy and tissue gastric biopsy. The bacteria is eliminated with the stools what would permit diagnose carrying out PCR in the samples of patients before and during the treatment. However, the stools contain inhibitors that could difficult the development of PCR in the samples. Here we have compared methods of DNA extraction of stools based in a boiling samples and described before for Holland (2000), with another method alkaline lyses modificated with use of Triton X-100 improving of DNA extraction. DNAs from stools samples of 10 patients were used to PCR reaction with specific primers described before to Helicobacter pylori (urec, RNAr e vac), and 5 samples were amplificated the specific manner, coincident with the Giemsa (+) tissue gastric diagnostic from dyspepsia patients. L. chagasi protozoa is responsible of Visceral leishmanoses with a high incident and distribution in Brazil, and is lethal when associated at to bad nutrition and opportunistic infections. The diagnostic is carried out mainly by the visualization of the parasite in the spleen, liver, bone marrow or lymphatic aspirates. In the present work we development a simple PCR using different DNAs from tripanosomatideos parasites and PIA3 and DEB8 specific primers to L. chagasi. The two primers were specific to L. chagasi, and the sensibility to PIA3 was 10 pg and for DEB8 was 1 pg. Multiplex PCR was development with the objective to use to Visceral and Tegumentary leishmanioses diagnostic differentiation, for this was used a specific primer to Viannia subgenera named LV1 and primers specific to L. chagasi. The fourth primers did not have any competition between them in the reaction and had the same specificity and sensibility to the single PCR. After bioinformatic analyze of Trypanosome Sanger Gene data banks was choosen an orphan gene of the L. infantum to use for L. chagasi diagnostic. 7.078 hypothetical proteins of L. infantum were compared with 11.812 of T. cruzi, 7557 of T. brucei and 5.364 of L. major. Using Omni blast and Protogim program was possible selected 93 hypothetical proteins of L. infantum. Based on size, cellular localization and others criteriaamp;#8217;s, 5 putatives primer were choosen to use in vitro assays. A Linj 20074 was highly specific and with 1pg of sensibility. Orphan genes could be promissory alternative to diagnostic or target quimioterapico in parasites illness
This document abstract is also available in Portuguese.
Bibliographical Information:

Advisor:Paulo Paes de Andrade

School:Universidade Federal de Pernambuco

School Location:Brazil

Source Type:Master's Thesis

Keywords:Molecular Biology Leishmania chagasi Helicobacter pylori


Date of Publication:03/10/2006

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