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

      Experimental Parasitology
      Animals, Base Sequence, Cysteine Proteases, genetics, Deoxyribonucleases, Type II Site-Specific, Dogs, Humans, Insect Vectors, Leishmania donovani, enzymology, isolation & purification, Leishmania infantum, Molecular Sequence Data, Polymerase Chain Reaction, Polymorphism, Restriction Fragment Length, Psychodidae, Restriction Mapping, Species Specificity

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          Abstract

          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.

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