Multilocus Microsatellite Polymorphism Analysis to Characterize Leishmania infantum Strains Isolated in Sicily

Abstract

Different approaches are being developed to improve the differentiation of Leishmania genus using biochemical and molecular methods. In this study, 11 independent polymorphic microsatellites were used for the typing of strains of L. infantum isolated in Sicily. Polymerase chain reaction was employed to amplify the microsatellites contained in 12 DNA regions selected from among more investigated loci. A total of 51 isolates of L. infantum from dogs were tested by using the same locus panel. The products were successively analysed using an automatic sequence detector (ABI PRISM 3130 AB), to discover relevant microsatellite polymorphisms. It was possible to discriminate between MON-1 and non-MON-1 groups. Moreover, the method permitted to distinguish various genotypes of L. infantum isolates within each zymodema. Model- and distance-based analyses of the data set showed comparable results. The frequency of heterozygosity in the alleles analysed varied extremely between the different groups of isolates. As the method exhibits a high level of discrimination, it is suitable for characterization of closely related strains in epidemiological studies.

Genetic diversity and structure in Leishmania infantum populations from southeastern Europe revealed by microsatellite analysis

“…As shown in Table 2, the highest allelic richness, expected and observed heterozygosities were demonstrated for markers Lm4TA, Lm2TG and Li22-35 (E), with the expected heterozygosity (mean H e  = 0.29) always being much higher than the observed (mean H o  = 0.02). Inbreeding coefficients (F IS ) were substantially high (mean F IS  = 0.92), as four markers presented absolute heterozygote deficiency (F IS  = 1), and marker Li46-67 (C) was monomorphic as previously observed [52,54]. …”

Multilocus microsatellite typing (MLMT) reveals host-related population structure in Leishmania infantum from northeastern Italy

“…donovani complex [ 12 , 13 , 15 ], were investigated in the present study. The amplification reaction of the microsatellite loci was performed as described elsewhere [ 22 ], using fluorescent-labeled forward primers (WellRed dyes, Sigma-Aldrich, Saint Louis, USA), and employing the described annealing temperature (Ta) except for loci LIST7039 (Ta = 54°C), CS20 (Ta = 56°C) and LIST7031 (Ta = 54°C). The amplicons were subjected to automated fragment analysis on the capillary CEQ 8000 sequencer (Beckman Coulter Inc., Indianapolis, USA) and the fragment sizes were determined using the “Fragment” package of the CEQ 8000 version 8.0 software.…”

Serological and entomological survey of canine leishmaniasis in Lampedusa island, Italy

“…DNA was extracted from lymph node aspirate samples using a Invitrogen PureLink Genomic DNA Mini Kit according to manufacturer’s instructions. The PCR test was targeted to a 68 bp fragment inside the constant region of kinetoplast DNA (kDNA) (NCBI accession number AF291093) as previously described [ 9 ]. The primer sequences were: QLK2-U 5′-GGCGTTCTGCGAAAACCG-3′; QLK2-D5′-AAAATGGCATTTTCGGGCC-3′; probe: 5′-TGGGTGCAGAAATCCCGTTCA-3′ 5′FAM with 3′ BHQ labelled.…”