Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples

Ibrahim Abbasi, Oscar D. Kirstein, Asrat Hailu, Alon Warburg*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

Abstract

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 0.1 pg 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.

Original languageAmerican English
Pages (from-to)20-26
Number of pages7
JournalActa Tropica
Volume162
DOIs
StatePublished - 1 Oct 2016

Bibliographical note

Funding Information:
This study was supported by the Bill and Melinda Gates Foundation Global Health Program (grant number OPPGH5336 ).

Publisher Copyright:
© 2016 The Authors

Keywords

  • Isothermal loop-mediated amplification
  • Leishmania donovani
  • Real time LAMP
  • SYTO16
  • Visceral leishmaniasis

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