Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides (original) (raw)
Related papers
PubMed, 2021
Background: Leishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome. Method: In the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions. Results: The lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes). Conclusion: In conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites.
Experimental Parasitology, 2011
Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.
Journal of clinical microbiology, 1994
The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy ...
FEMS Microbiology Letters, 1996
We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Laishmunia strain UR6 with other minicircle sequences from different Leishmaniu species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.
Single-Step Multiplex PCR Assay for Characterization of New World Leishmania Complexes
1989
We have developed a PCR assay for one-step differentiation of the three complexes of New World Leishmania (Leishmania braziliensis, Leishmania mexicana, and Leishmania donovani). This multiplex assay is targeted to the spliced leader RNA (mini-exon) gene repeats of these organisms and can detect all three complexes simultaneously, generating differently sized products for each complex. The assay is specific to the Leishmania genus and does not recognize related kinetoplastid protozoa, such as Trypanosoma cruzi, Trypanosoma brucei, and Crithidia fasciculata. It correctly identified Leishmania species with a broad geographic distribution in Central and South America. The sensitivity of the PCR amplification ranged from 1 fg to 10 pg of DNA (0.01 to 100 parasites), depending on the complex detected. Crude extracts of cultured parasites, prepared simply by boiling diluted cultures, served as excellent templates for amplification. Crude preparations of clinical material were also tested. The assay detected L. braziliensis in dermal scrapings from cutaneous leishmanial lesions, Leishmania chagasi in dermal scrapings of atypical cutaneous leishmaniasis, and L. mexicana from lesion aspirates from infected hamsters. We have minimized the material requirements and maximized the simplicity, rapidity, and informative content of this assay to render it suitable for use in laboratories in countries where leishmaniasis is endemic. This assay should be useful for rapid in-country identification of Leishmania parasites, particularly where different Leishmania complexes are found in the same geographical area.
Un ensayo de PCR para identificar especies de Leishmania del subgénero Viannia
Rev. Soc. Ven. Microbiol, 2011
We have identified a novel DNA sequence of 500 bp (β500-DNA) on the Leishmania (Viannia) subgenus, located in the intergenic region of one of the loci of the β-tubulin gene family. The sequence analysis showed that this sequence has no homology to any other sequence described so far, including the β-tubulin gene. We improved a specific β500-PCR assay, which generated a PCR product of 375 bp for total genomic DNA from Leishmania strains belonging to the L. (Viannia) subgenus. In contrast, no amplification was found when using genomic DNA from species of L. (Leishmania) subgenus or other organisms. Under our PCR conditions, the lower detection limit was 1 fg when a purified DNA clone (pLgβ4), which contains one copy of the β500-DNA sequence, was used. The β500-DNA PCR assay confirmed the preliminary diagnosis of cutaneous leishmaniasis in clinical samples in which the Montenegro skin test was positive and parasite cultures were negative. The analytical specificity and the sensitivity of the PCR assay provide a tool for epidemiological studies of the disease.
Experimental Parasitology, 2001
RAPD fragments studied correspond to mechanisms already described. Although they do not account for the amplification of all Leishmania analyses of old world leishmania RAPD markers and development of a PCR assay selective for parasites of the L. donovani species complex. RAPD products, such mechanisms stress some of the pitfalls of the technique, which need to be taken into consideration. We have identified Experimental Parasitology 98, 90-99. Three amplicons, appearing in a species-specific manner on the electrophoregrams of RAPD reactions at least misleading observations of DNA bands amplified in a speciesspecific manner, in spite of their presence in the genome of the other that were obtained with primer OPA1, OPA1-800, OPA1-900, and OPA1-1200, are analyzed in this study. The study revealed that each taxa, and relatedness between bands within the amplification profiles. Therefore, recommendations for careful interpretation of RAPD data in of these products is composed of one Leishmania DNA band, taxonomically conserved among the different Old World species studied. Subse-population genetics or phylogenetic analyses are reiterated. Molecular analyses are essential to validate conclusions. ᭧ 2001 Academic Press quently, only the electrophoretic position of the RAPD products can be considered species-specific. In addition, sequence data, genomic Index Descriptors and Abbreviations: Leishmania donovani species complex; Old World Leishmania; visceral leishmaniasis (VL); cutane-organization, and chromosomal location have proved that these fragments are different and physically independent. However, they possess ous leishmaniasis (VL); canine leishmaniasis (CanL); RAPD (random amplification of polymorphic DNA); decamer primer; amplicon analy-common features related to the presence of different kinds of short DNA repeats, more particularly microsatellites and a CCCTTC motive, sis; sequence features; molecular mechanisms; base content; GC%; DNA marker; probe; parasite discrimination; RFLP (restriction frag-corresponding to the 3Ј half of the OPA1 primer. These results suggest that the OPA1 primer has initiated amplification from different priming ment length polymorphism); PCR (polymerase chain reaction); selective PCR for L. donovani complex; PCR-RFLP assay; molecular epide-sites, having a species-specific location. This corresponds to sequence micro-heterogeneity of DNA fragments present within the different miology; microsatellites; bp (base pairs). species and leading eventually to a selective amplification of different RAPD products. This characteristic has been used to develop an original selective PCR test based on the sequence of the OPA1-800 product, in which only DNAs from the L. donovani species complex are amplified. Restriction site polymorphisms and sequence variations are identified within the PCR fragment amplified from these parasite DNAs. In fact,