Evaluation and improvement of two PCR targets in molecular typing of clinical samples of Leishmania patients.
Leishmaniasis is a disease caused by the unicellular Leishmania parasite. World wide millions of people are affected by this vector born disease. The disease presents itself in different clinical manifestations which are caused by specific Leishmania species. The therapeutic strategy depends on the Leishmania species involved. It is important to detect Leishmania and subsequently type the infecting species in a sensitive way using PCR. Various targets have been proposed but two seem to be best suited, the ITS1 region and the mini-exon. There is, however, no consensus as to which of these two is best. The aim of this study was to compare both targets with our current method, a PCR on the 18S ribosomal RNA gene. The ITS1 PCR proved to be slightly more sensitive and more practical than the mini-exon. Nevertheless, the mini-exon is more polymorphic and is needed in subtyping Leishmania species belonging to the L. Viannia subgenus. The ITS1 method was adapted to use as a real-time PCR for diagnostic purposes. In addition, designing and testing a new primer set improved sensitivity of the PCR on the mini-exon.
Roelfsema
Jeroen H
JH
National Institute of Public Health and the Environment, Centre for Infectious Disease Control Netherlands, The Netherlands. jeroen.roelfsema@rivm.nl
Nozari
Nahid
N
Herremans
Tineke
T
Kortbeek
Laetitia M
LM
Pinelli
Elena
E
D001853:Bone Marrow / Q000469:parasitology; D016054:DNA, Protozoan / Q000032:analysis* / Q000737:chemistry; D004275:DNA, Ribosomal / Q000032:analysis / Q000737:chemistry; D005091:Exons / Q000235:genetics; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis / Q000469:parasitology*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000469:parasitology*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D011446:Prospective Studies; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012340:RNA, Ribosomal, 5.8S / Q000235:genetics; D015183:Restriction Mapping; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012867:Skin / Q000469:parasitology
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D012340:RNA, Ribosomal, 5.8S