A Novel Non-Amplification Assay Detects Leishmania

The need for accurate diagnostic investigation of cases of leishmaniasis has made the use of molecular biology techniques broadly applicable for identifying the protozoan parasite.

The reliable application of these methods requires highly specialized personnel, dedicated equipment and space and because of these drawbacks of the molecular diagnostic approach, has targeted several alternatives and the most recent of which is the incorporation of nanoparticles for the detection of specific bio-molecules.

Laboratory scientists at the University of Athens (Greece) used for the assessment of the method’s specificity 15 cultured isolates of various Leishmania species from human and animal origin. There were 40 negative controls selected among pathogens genetically related to Leishmania spp., commonly found in clinical samples, both human and canine, as well as some bacterial species.

DNA isolation was performed on all samples by the Nucleospin Tissue kit (Macherey Nagel; Düren, Germany). Gold nanoparticles (AuNPs, Nanopartz; Salt Lake City, UT, USA) were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. The results were recorded by visual observation of the test tubes or by spectrophotometry (Infinite M200, Tecan; Männedorf Switzerland). In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red.

The assay produced consistently positive results detectable by simple visual observation of the test tubes with no less than 115 ng of leishmanial DNA in a sample volume of 10 μL and therefore the method's minimum detection limit was defined as 11.5 ng of target DNA per μL of sample. Repeatability and reproducibility were 100% and relative sensitivity and specificity referenced to a classical polymerase chain reaction (PCR) method were calculated to 92% and 100% regarding collectively control and clinical samples.

The authors concluded that the method provides an easy, fast, and inexpensive way for Leishmania DNA detection using AuNPs conjugated to four single strand oligonucleotide-probes. This approach can be considered an appealing diagnostic solution for resource-poor settings, especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not a top priority. The study was published in the January 2014 issue of the Journal of Microbiological Methods.

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University of Athens
Macherey Nagel
Tecan