NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

Abstract

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis,Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis,Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

“…Santamaria et al (2005) determined the LoD in 1 equivalent parasite in the PCR using kDNA (Santamaria et al, 2005), unlike the findings of this study, the LoD of this technique with the same marker was 1 × 10 -1 parasites equivalents/mL ( Figure 1 ). These results are consistent with those described by other authors where is reported that kDNA presents high copy number and can detect up to less than one parasite in conventional PCR (de Bruijn and Barker, 1992; Mary et al, 2004; Francino et al, 2006; Oliveira et al, 2011; Gualda et al, 2015). The LoD for the assays directed to the 18S and ITS-1 markers was 1 × 10 0 parasites equivalents/mL, similar results reported by Deborggraeve et al (2008) where the technique was able to detect up to 1 parasite/180 μL.…”

Canine Leishmaniosis Control through the Promotion of Preventive Measures Appropriately Adopted by Citizens

“…For DNA extraction, the NucleoSpin® Tissue Kit (Macherey-Nagel, GmbH & Co. KG, Germany) was used according to the manufacturer's instruction. Then, the extracted DNA was amplified using a SYBR® Green Real-Time PCR targeting a kDNA gene (230 bp) with the forward primer FLC2 (5′-GTCAGTGTCGGAAACTAATCCGC-3′) and the reverse primer RLC2 (5′-GGGAAATTGGCCTCCCTGAG-3′) designed by Gualda et al [ 22 ]. The cycling conditions, performed in a 7900HT thermocycler (Applied Biosystems, California, USA), were as follows: the final reaction volume was 20 μ l with 10 μ l QuantiFast™ SYBR® Green PCR Master Mix 2x concentrated (Qiagen, Germany), 6.6 μ l RNase-free water, 0.2 μ l (0.1 μ mol) of each specific primer, and 3 μ l of the DNA isolated from the blood samples.…”

Asymptomatic Leishmania infection in blood donors from a major blood bank in Northeastern Brazil: a cross-sectional study

“…Positive samples by qPCR were further tested by a species-specific conventional PCR assay, using the primers FLC2 (5'-GTCAGTGTCGGAAACTAATCC GC-3') and RLC2 (5'-GGGAAATTGGCCTCCCTGAG-3'), which amplify a 230 bp fragment of L. infantum kDNA 13 . The final reaction volume was 25 μL, consisting of 1 X buffer, 0.2 mM dNTP (dATP, dCTP, dGTP, and dTTP), 1.5 mM MgCl 2 , 1.5 U Taq Polymerase (Invitrogen,Thermo Scientific, Waltham, MA, USA), 0.4 pmol of each primer and 2 μL of genomic DNA.…”