Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.
Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR.
Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Israel; Department of Biological Sciences, Faculty of Science and Technology, Al-Quds University, Abu Deis, the Palestinian Territories. Electronic address: ibrahima@ekmd.huji.ac.il.
Ibrahim
I
Abbasi
Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Israel. Electronic address: oscark@ekmd.huji.ac.il.
Oscar D
OD
Kirstein
Department of Microbiology, Immunology & Parasitology, Faculty of Medicine, Addis Ababa University, Addis Ababa, Ethiopia. Electronic address: hailu_a2004@yahoo.com.
Asrat
A
Hailu
Department of Microbiology and Molecular Genetics, The Institute for Medical Research Israel-Canada, The Kuvin Centre for the Study of Infectious and Tropical Diseases, The Hebrew University - Hadassah Medical School, The Hebrew University of Jerusalem, 91120, Israel. Electronic address: alonw@ekmd.huji.ac.il.
Alon
A
Warburg
D000328:Adult; D000351:Africa, Eastern; D000368:Aged; D000369:Aged, 80 and over; D001681:Biological Assay; D001769:Blood / Q000469:parasitology*; D001938:Brazil; D017931:DNA Primers / Q000235:genetics*; D018105:DNA, Kinetoplast / Q000235:genetics; D016054:DNA, Protozoan / Q000097:blood*; D005260:Female; D006801:Humans; D007893:Leishmania donovani / Q000235:genetics* / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000097:blood* / Q000469:parasitology*; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D018105:DNA, Kinetoplast; D016054:DNA, Protozoan
Isothermal loop-mediated amplification; Leishmania donovani; Real time LAMP; SYTO16; Visceral leishmaniasis