RESUMEN
Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/genética , Leptospira/clasificación , Leptospira/genética , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis por Conglomerados , Enzimas de Restricción del ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Genotipo , Leptospira/aislamiento & purificación , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , SerogrupoRESUMEN
Estudou-se 96 amostras de carnes de räs procedentes de abatedouros sob Inspeçäo Sanitária e sem Inspeçäo Sanitária de diferentes estados do Brasil, com isolamento de 61 bolores e 240 leveduras.As leveduras foram identificadas em 10 gêneros diferentes, distribuídos em 28 espécies.Resalta-se a ocorrência de Canddida albicans e Candida tropicalis pelo potencial patogênico e por näo serem de ocorrência comum em carnese derivados.Osresultados indicam que a microbiota fúngica em carnes de räs é predominantemente composta de leveduras isoladas de acordo com os tipos de abatedouros estudados näo foram, estatisticamente, significativas.