Discrimination Amongst Leishmania by Polymerase Chain Reaction and Hybridization with Small Subunit Ribosomal DNA Derived Oligonucleotides

Abstract

A method for discriminating among Leishmania is described, based upon small subunit ribosomal DNA sequence differences. The method was to amplify the entire 2.2 kb small subunit rDNA by polymerase chain reaction using conserved primers specific for the 5' and 3' termini of the small subunit ribosomal RNA, and then hybridize the product dotted onto nylon membranes with labeled oligonucleotides. The design of the hybridization probes was based upon complete small subunit rDNA sequences from L. amazonensis, L. major and L. guyanensis and partial sequences of L. mexicana, L. braziliensis, L. tropica and L. chagasi. A high degree of sequence similarity (> 99%) among species was found. However, sufficient sequence divergence occurred to permit the design of internal oligonucleotide probes specific for species complexes. This procedure successfully discriminated amongst a wide range of Leishmania isolates. The method detected as few as 10 cultured organisms and detected parasites in tissue samples from experimentally infected animals. Non-radioactive labeling showed the same specificity and sensitivity as radioactive probes.

Ocurrence of co-infection by Leishmania (Leishmania) chagasi and Trypanosoma (Trypanozoon) evansi in a dog in the state of Mato Grosso do Sul, Brazil

“…Reactions were performed in a final volume of 50 µl containing 1X PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTPs, 0.2 µM of each primer and 2 U Taq DNA polymerase. The first PCR was performed with primers S4 (5'-GAT CCA GCT GCA GGT TCA CC -3') and S12 (5'-GGT TGA TTC CGT CAA CGG AC -3') as described by Uliana et al (1994). The DNA was denatured at 94ºC for 3 min and then cycled 35 times at 94ºC for 1 min, at 50ºC for 1 min, and at 72ºC for 1 min.…”

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

“…Another advantage of this technique is that it requires low parasite density, i.e. a minimum level of 10³ parasites/mL 20 . This same author has reported that given the high sensitivity of the test, it is possible to perform PCR with just 10 parasites and to detect the band in the electrophoresis gel.…”

“…testing for Leishmania in the aspirate of bone marrow, lymph nodes, blood or skin, is an invasive method 5 that calls for specialist knowledge and thus is not practicable when there is a high demand for these exams. To resolve this dilemma, the PCR technique offers a solution by means of specific primers that make identification of the various Leishmania species possible 19,20,22 . Another advantage of this technique is that it requires low parasite density, i.e.…”

Natural infection of phlebotomines (Diptera: Psychodidae) in a visceral-leishmaniasis focus in Mato Grosso do Sul, Brazil

“…After DNA extraction, two PCR methodologies were used. The first (the initial screening) identified trypanosomatids by means of primers that amplify ribosomal DNA genes (rDNA) 20 . The second methodology, which used primers that were complementary to the miniexon sequences, enabled the Leishmania 16 species among the trypanosomatids to be identified.…”