Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB).
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.
Universidade Federal de Minas Gerais, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Lenice Roteia Cardoso
LR
Jung
Universidade Federal de Minas Gerais, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. ; Universidade do Ceuma, Departamento de Biologia Parasitária, Universidade Ceuma, São Luis, MA, Brasil, Universidade Ceuma, Departamento de Biologia Parasitária, São Luis, MA, Brazil.
Maria Rosa Quaresma
MR
Bomfim
Universidade Federal de Minas Gerais, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil, Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Erna Geessien
EG
Kroon
Universidade Federal de Minas Gerais, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
Álvaro Cantini
ÁC
Nunes
D016000:Cluster Analysis; D004262:DNA Restriction Enzymes / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D007919:Leptospira / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D065288:Serogroup
D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D004262:DNA Restriction Enzymes
DNA typing; Leptospira; RFLP; rpoB gene; serovar