engleski

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism (RFLP), real-time PCR and pulsed-filed gel electrophoresis (PFGE)

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analyzed. For serovar identification, microscopic agglutination test (MAT) and pulsed field gel electrophoresis (PFGE) were used. In order to identify isolates on genomic species level, polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the serogroup affinities as following: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331 bp long fragment of rrs (16S rDNA gene) digested with endonucleases MnlI and DdeI and the real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to L. kirschneri, Icterohaemorrhagiae isolates to L. interrogans and Javanica isolates to L. borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analyzed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate diversity of leptospires circulating in Croatia. At the same time, we point out on the usefulness of combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Leptospira; leptospirosis; RFLP; PFGE; Real-Time PCR

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