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- IOC - Artigos de Periódicos [12678]
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SENSITIVE DETECTION AND SCHIZODEME CLASSIFICATION OF TRYPANOSOMA CRUZI CELLS BY AMPLIFICATION OF KINETOPLAST MINICIRCLE DNA SEQUENCES: USE IN DIAGNOSIS OF CHAGAS' DISEASE
Chagas Disease
Polymerase chain reaction
Minirepeat
Minicircle DNA
Schizodeme
Afiliação
University of California. Department of Biology and Molecular Biology Institute. Los Angeles, CA, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil
University of California. Department of Biology and Molecular Biology Institute. Los Angeles, CA, USA.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.
Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil
University of California. Department of Biology and Molecular Biology Institute. Los Angeles, CA, USA.
Resumo em Inglês
Amplification of DNA sequences from the kinetoplast minicircle DNA was employed as a method for the detection and classification
of small numbers of Trypanosoma cruzi cells. Two overlapping fragments from the conserved 120 bp minirepeat regions
of the minicircle DNA and one fragment covering the adjacent variable regions were amplified. The minimal amount of minicircle
DNA required to detect a product by hybridization with an oligonucleotide probe was 0.015 fg. which represents approximately
10 molecules or 0.1% of the minicircle DNA component of a single cell. The amplification worked equally well with kDNA from
several strains of T. cruzi and did not occur with kDNA from several other kinetoplastids. kDNA recovered from less than 10
trypanosomes in whole blood could be used as a template for amplification; the presence of a several billion fold excess of human
DNA had no effect on the amplification process. Schizodeme analysis by hybridization with specific oligonucleotides or by direct
restriction enzyme digestion could be performed on the amplified fragments representing the minicircle conserved region or variable
regions. This method should prove useful as a rapid, specific and sensitive assay for Chagas' disease in chronic patients as
well as for epidemiological studies of infected animals and insects.
Palavras-chave em inglês
Trypanosoma cruziChagas Disease
Polymerase chain reaction
Minirepeat
Minicircle DNA
Schizodeme
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