Inter-simple-sequence repeat (ISSR)-PCR for the identification of saprophytic strains of Leptospira

Abstract

The inter-simple-sequence repeat (ISSR) primers that anneal to a simple repeat of various length and at non-repetitive motifs at 3′ and 5′ end were attempted for PCR amplification of Leptospira genome. Of the six ISSR primers tested, namely, (AG)8T, (AG)8C, (AG)8G, (CA)8A, (TG)8C and (TG)8G, only primer (AG)8T produced amplification of 1000 bp in the two non-pathogenic Leptospira species tested, viz; Leptospira biflexa serovar patoc and L. meyeri serovar ranarum, with no amplification in any of the 16 standard pathogenic serovars tested. The remaining five ISSR primers did not exhibit any amplification of the Leptospira genome in either pathogenic or non-pathogenic species. From among 35 Leptospira isolates recovered from hospitalized patients with pyrexia of unknown origin and/or febrile jaundice (12 in number) and from different environmental water sources (23 in number), (AG)8T ISSR-PCR correctly identified all the 22 isolates from water sources that were confirmed to be non-pathogenic by conventional tests. The results therefore, confirmed the ability of a primer, based on simple-sequence repeat motif, to produce a fragment that is useful as a group genetic marker in Leptospira species. The single nucleotide anchor, T′, at the 3′ end of the primer appeared to play an important role in differentiation of pathogenic and non-pathogenic species of Leptospira. Multiplex PCR, using ISSR primer, (AG)8T and the reported 16S rRNA gene primers, specific for pathogenic Leptospira species, or the 23S rRNA Leptospira genus specific primers, provided clear identification of serovars and isolates into pathogenic or non-pathogenic groups.