Enzymatic amplification of mini-exon-derived RNA gene spacers of Leishmania donovani: primers and probes for DNA diagnosis

Abstract

SUMMARY: The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania donovani was enzymatically amplified by the polymerase chain reaction (PCR). The major 180 bp PCR product contained conserved med RNA gene sequences flanking the variable intergenic spacer from the med RNA gene tandem repeat. The oligonucleotide primers cross-reacted with other Leishmania species. In serial dilution experiments, positivity in the PCR assay was observed down to the genomic DNA equivalent of less than a single Leishmania cell. When the major PCR products from Indian L. donovani isolates were cloned and used as probes in dot hybridization analyses, they discriminated between L. donovani and L. amazonensis, L. major and L. infantum under high stringency conditions. DNA from spleen biopsies and blood samples of confirmed kala azar patients was positive, as were two skin biopsies from patients with post-kala azar dermal leishmaniasis (PKDL). These observations demonstrate that PCR amplification of med RNA intergenic spacers is sufficiently sensitive for clinical diagnosis of kala azar and PKDL, and furthermore, that cloned intergenic spacer probes may be useful for identification and classification of L. donovani.