Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)
Braz. j. microbiol
; Braz. j. microbiol;46(2): 465-476, Apr-Jun/2015. tab, graf
Article
en En
| LILACS
| ID: lil-749718
Biblioteca responsable:
BR1.1
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
LILACS
Asunto principal:
Polimorfismo de Longitud del Fragmento de Restricción
/
ARN Polimerasas Dirigidas por ADN
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Reacción en Cadena de la Polimerasa
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Técnicas de Diagnóstico Molecular
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Leptospira
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Braz. j. microbiol
Asunto de la revista:
MICROBIOLOGIA
Año:
2015
Tipo del documento:
Article
País de afiliación:
Brasil
Pais de publicación:
Brasil