Detection and identification of pathogenic leptospires in bio-meterials

Abstract

In the present study an attempt has been made to detect and differentiate leptospires in bio-materials employing molecular techniques such as genus specific PCR, multiplex PCR, nested PCR, AP-PCR, LS-PCR and PCR-REA. Isolation trials for Leptospira were also made in the study and the isolates were tried to be differentiated employing the molecular methods mentioned above. The genus specific primers A and B were used to detect leptospires in clinical samples and samples from rodents. Out of the 147 samples only nine were positive for leptospiral DNA. Out of the nine positive samples eight were serum samples (four from cattle and four from dogs) and one was kidney of a bandicoot. Multiplex PCR, using the primers G1/G2 and B64-I/B64-II could differentiate leptospires into pathogenic and non-pathogenic ones. Among the pathogenic leptospires it could differentiate the species L. kirschneri, from other six pathogenic species viz L . interrogans, L. borgpetersenii, L. santarosai, L. noguchii, L. inadai and L. weillii. All the six isolates were found to be belonging to any of these six species. Nested PCR using the primers designed based on the sequence of L. borgpetersenii and L. interrogans amplified DNA from all the ten reference strains including the non-pathogenic serovar patoc and rachmati of L. kirschneri species. All the six isolates were amplified giving PCR products of expected sizes, 571 bp and 370 bp.