Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L-infantum/L-donovani discrimination

Discrimination of Leishmania infantum and L donovani, the members of the L (L) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA(PCR-RFLP) and a PCR that are capable to discriminate L donovani from L infantum. Typing of the complex was performed by a simple PCR of cysteine protease B (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3' end of cpb gene of L. donovani whereas it does not generate an amplicon for L infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci. (C) 2009 Elsevier Inc. All rights reserved.