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

Abstract

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.

Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity

“…Other repetitive molecular targets do not present this inconvenience and could lead to better accuracy, but their sensitivities are too low for survey and epidemiologic purposes. Hassan et al (10) proposed the use of mini-exon-derived RNA genes, present at 200 to 250 copies per cell, to improve sensitivity to 0.1 Leishmania parasites after amplification, electrophoresis, and hybridization; this technique was found to be 10-fold less sensitive than a kinetoplast DNA-based method. Piarroux et al (24) detected Leishmania DNA by targeting a repeated genomic sequence, and this method reached a sensitivity of 1 parasite per reaction; this assay was sufficient for the diagnosis of the disease but not for survey after therapy.…”

Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan

“…As a result, much effort has been directed towards the development of new and more sensitive diagnostic techniques. molecular biology is the polymerase chain reaction (PCR; Saiki et al 1988 Hassan et al 1993) or repetitive nuclear sequences (Piarroux et a[. 1993 diagnosis of L. donovani infections using blood, bone marrow and lymph node samples from Sudanese patients with parasitologically confirmed infection.…”

Quantitative Real-Time PCR Is Not More Sensitive than “Conventional” PCR

“…Thus, an assay using suboptimal PCR primers that target this extremely repetitive target may not reach the sensitivity of a more classical PCR that targets a moderately repetitive gene. Conversely, several PCR assays targeting the Ͼ100-fold-reiterated spliced leader RNA gene in Leishmania have consistently shown a mediocre analytical sensitivity compared with those targeting the Ͻ20-fold-repeated ribosomal DNA (about 1 and 100 parasites, respectively, detected per reaction) (4,17,21,22,29). Still, in the majority of cases, the degree of reiteration of the target is proportional to the sensitivity of the assay (i.e., the more copies of the target are present, the easier it is to detect).…”