Resumo

ALEGRE, E.A et al. Detección y diferenciación molecular de Leptospira sp. utilizando diferentes técnicas de extracción de ADN. Rev. vet. [online]. 2013, vol.24, n.1, pp.53-55. ISSN 1669-6840.

There are different diagnostic techniques to identify leptospiras; the polymerase chain reaction (PCR) is a method with high sensitivity and specificity. This study aimed to optimize DNA extraction from bacterial cultures and set conditions of multiplex PCR to differentiate saprophytes from pathogens leptospiras. For this, two techniques were tested: cetyl trimethyl ammonium bromide (CTAB) and boiling. Two sequences were analyzed, corresponding to a fraction of a conserved gene in both groups of bacteria (16S rRNA) and another in a fraction of a gene present only in the pathogenic leptospiras (LipL32). Two amplicons of 474 and 430 pb respectively were obtained using cultures of Leptospira icteroahemorragiae, L. canicola and apathogenic (Patoc I) as positive controls and water as negative control. It was concluded that there was no difference in the durability of DNA extracted from reference strains compare with CTAB digestion and boiling and its variants. Considering its lower cost and time saving, the latter represents a good alternative for PCR amplification.

Palavras-chave : Leptospira sp; Molecular differentiation; DNA extraction; Boiling.

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