[Comparative attempts for the establishment and optimisation of a PCR on Leishmania for the purpose of diagnosis].
Leishmania spp. are the causative agents of visceral, cutaneous and mucocutaneous leishmaniosis with several taxa ("species") of the genus Leishmania being involved in human disease. As diagnostics based on microscopical detection of the parasites or on serological tests are often unsatisfactory, also molecular biological methods, particularly the polymerase chain reaction (PCR), have been employed for the detection of Leishmania spp. in the past years. The aim of the present study was to compare different PCR-protocols and optimise them for our needs, placing emphasis on the improvement of DNA isolation. PCR was performed with whole cell DNA isolated from cultures, as well as from simulated blood samples and clinical samples. Three different methods for the isolation of DNA from blood samples and two different PCR-protocols, one amplifying a fragment of the 18S rDNA and one for the amplification of the whole kDNA-circle, were applied and compared. No significant difference in sensitivity was detected between the different PCR-protocols, however, it was shown that the highest yield of DNA was achieved with a DNA isolation protocol based on urea.
Fürnkranz
Ursula
U
Abteilung für Medizinische Parasitologie, Klinisches Institut für Hygiene und Medizinische Mikrobiologie der Medizinischen Universität Wien, Wien, Osterreich.
Walochnik
Julia
J
Grimm
Felix
F
Deplazes
Peter
P
Aspöck
Horst
H
D000818:Animals; D017931:DNA Primers; D018105:DNA, Kinetoplast / Q000302:isolation & purification; D016054:DNA, Protozoan / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000302:isolation & purification; D018314:Leishmania infantum / Q000235:genetics* / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000175:diagnosis* / Q000469:parasitology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D004275:DNA, Ribosomal