Leishmania: Identification of Old World Species Using a Permissively Primed Intergenic Polymorphic-polymerase Chain Reaction

We have developed a permissively primed intergenic polymorphic-polymerase chain reaction (PPIP-PCR) which distinguishes between the Old World Leishmania complexes L. major, L. tropica, L. donovani, and L. aethiopica. This technique pairs one parasite-specific and one nonspecific oligonucleotide primer for the PCR. The specific primer was chosen from a unique leishmanial DNA sequence, clone pDOG 2, isolated from a L. donovani chagasi genomic DNA expression library. This sequence has a high DNA homology to the intergenic region of the L. major B/C genes which belong to the polymorphic LmcDNA16 gene family. The specific intergenic primer contains a high GC content, a stem-loop, and a 3'-CG residue. The nonspecific primer was selected from within the pBluescript (SK) plasmid. Using PPIP-PCR, parasites belonging to the L. major, L. tropica, L. donovani, and L. aethiopica complexes could be easily identified directly following agarose gel electrophoresis by the simple profiles of their PCR products. In addition, it was possible to discriminate between strains of L. major or L. donovani from distant geographical regions. Amplification of genomic DNA isolated from several nonleishmanial kinetoplastids yielded either no PCR products or unique bands which were distinct from the leishmanial profiles. Genomic DNA from nonkinetoplastid parasites, plants, or mammals was not amplified by PPIP-PCR. This technique is a rapid and reproducible method for the characterization of Old World Leishmania.