Use of molecular probes and PCR for detection and typing of Leishmania - a mini-review

Abstract

The use of molecular tools to detect and type Leishmania species in humans, reservoirs or sandflies has been pursued using different approaches. The polymerase chain reaction provided sensitivity to case this task, since the use of hybridization procedures alone employing specific probes is hampered due to the low detection limit. In this report, we describe the different molecular targets used in our laboratory, aiming at the detection and specific typing of these protozoa. Different kits based on hybridization assays and PCR amplification using kinetoplast and nuclear targets are described and the results obtained from their use are reported.

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

“…Two µl of DNA (10 ng/µl) were added per reaction and DNA amplification was performed as described, using primers previously designed for Leishmania spp. 7 : 5' GGG GAG GGG CGT TCT GCG AA 3'; 5'CCG CCC CTA TTT TAC ACC AAC CCC 3'; 5'GGC CCA CTA TAT TAC ACC AAC CCC 3'. In the case of amplification with specific primers, B1 (5' GGG GTT GGT GTA ATA TAG TGG 3') and B2 (5' CTA ATT GTG CAC GGG GAG G 3') for L. braziliensis 6 complex or M1(5'CCA GTT TCG AGC CCC GGA G3') and M2 (5'GGT GTA AAA TAG GGG CGG ATG CTC TG 3') for L. mexicana 8 complex, the following volumes were used: 5 µl of PCR buffer, 8 µl of dNTPs, 0.5 µl of each primer (20 pmol/µl), 2 µl of DNA (10 ng/µl), 0.5 µl de Taq DNA polymerase (2.5 U/µl) and 33.5 µl of ultrapure water.…”

Comparison of the specificity of PCR and the histopathological detection of leishmania for the diagnosis of American cutaneous leishmaniasis

“…PCR has been shown to be highly sensitive for the diagnosis of ACL, with rapid detection of leishmania (6)(7)(8)(9)(10)(11). PCR can also be used for the typing of leishmania isolated from clinical material, insect vectors, reservoirs, or culture media (12,13). The method showed 98.40% sensitivity and 95.59% specificity for the diagnosis of individuals from the municipality of Caratinga, Minas Gerais (14).…”

First encounter of subclinical human Leishmania (Viannia) infection in State of Rio Grande do Sul, Brazil

“…Addition- ally, another blood sample was collected and serum was separated for the detection of Leishmania antibodies by indirect immunofluorescence (IFI) and ELISA (Madeira et al 2000) and Trypanosoma cruzi by enzyme immunoassay and IFI . For PCR amplification, 1 ml of a 1:20 dilution of the DNA extracted from each sample was submitted to hot-start PCR with primers that amplify the conserved region of the minicircle molecules present in all Leishmania species (Degrave et al 1994). The reaction mixture contained 100 ng of each primer (5´-(G/C)(G/C)(C/G)CC(A/C)-CTAT(A/T)TTACACCAACCCC and 5´-GGGGAGGGG-CGTTCTGCGAA), 200 mM of each dNTP (GE Healthcare Life Sciences, São Paulo), 2.5 units of Taq polymerase (Ampliaq Gold, Perkin-Elmer, Norwalk) in the buffer supplied by the manufacturer, and 1.5 mM MgCl 2 .…”