ENZYMATIC AMPLIFICATION OF MINI-EXON-DERIVED RNA GENE SPACERS OF LEISHMANIA-DONOVANI - PRIMERS AND PROBES FOR DNA DIAGNOSIS

The multicopy mini-exon-derived RNA (med RNA) locus of Leishmania dono vani was enzymatically amplified by the polymerase chain reaction (PCR ). The major 180 bp PCR product contained conserved med RNA gene seque nces flanking the variable intergenic spacer from the med RNA gene tan dem repeat. The oligonucleotide primers cross-reacted with other Leish mania species. In serial dilution experiments, positivity in the PCR a ssay was observed down to the genomic DNA equivalent of less than a si ngle Leishmania cell. When the major PCR products from Indian L. donov ani isolates were cloned and used as probes in dot hybridization analy ses, they discriminated between L. donovani and L. amazonensis, L. maj or and L. infantum under high stringency conditions. DNA from spleen b iopsies and blood samples of confirmed kala azar patients was positive , as were two skin biopsies from patients with post-kala azar dermal l eishmaniasis (PKDL). These observations demonstrate that PCR amplifica tion of med RNA intergenic spacers is sufficiently sensitive for clini cal diagnosis of kala azar and PKDL, and furthermore, that cloned inte rgenic spacer probes may be useful for identification and classificati on of L. donovani.