title: A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles.
abstract: Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. 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 specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The method's minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.
authors.affiliation: Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens, 11855, Greece.
authors.forename: Margarita
authors.initials: M
authors.lastname: Andreadou
authors.affiliation: Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens, 11855, Greece.
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Liandris
authors.affiliation: Laboratory of Biology, School of Medicine, University of Athens, 176 Michalakopoulou, Athens, 11527, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Gazouli
authors.affiliation: Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens, 11855, Greece.
authors.forename: Styliani
authors.initials: S
authors.lastname: Taka
authors.affiliation: Faculty of Medicine, Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, University of Crete, Voutes, Heraklion, 71003, Crete, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Antoniou
authors.affiliation: Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens, 11855, Greece.
authors.forename: Georgios
authors.initials: G
authors.lastname: Theodoropoulos
authors.affiliation: Department of Medical Physics and Bioengineering, Biomedical Optics Research Laboratory, University College London, Gower Street, WC1E 6BT, London.
authors.forename: Ilias
authors.initials: I
authors.lastname: Tachtsidis
authors.affiliation: Department of Forensic Medicine and Toxicology, Medical School of Athens, 75M. Asias, Goudi, Athens, 11527, Greece.
authors.forename: Nikolaοs
authors.initials: N
authors.lastname: Goutas
authors.affiliation: Department of Forensic Medicine and Toxicology, Medical School of Athens, 75M. Asias, Goudi, Athens, 11527, Greece.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Vlachodimitropoulos
authors.affiliation: Bioinformatics & Medical Informatics Team, Biomedical Research Foundation, Academy of Athens, Soranou Efessiou 4, Athens, 11527, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Kasampalidis
authors.affiliation: Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science and Hydrobiology, Agricultural University of Athens, 75 Iera Odos, Votanikos, Athens, 11855, Greece. Electronic address: ikonomop@aua.gr.
authors.forename: John
authors.initials: J
authors.lastname: Ikonomopoulos
mesh_terms: D000818:Animals; D003116:Color; D018105:DNA, Kinetoplast / Q000032:analysis / Q000235:genetics; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004285:Dogs; D006046:Gold; D006801:Humans; D007891:Leishmania / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis* / Q000469:parasitology / Q000662:veterinary; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D053758:Nanoparticles; D015345:Oligonucleotide Probes / Q000235:genetics; D010274:Parasitology / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D018105:DNA, Kinetoplast; D016054:DNA, Protozoan; D015345:Oligonucleotide Probes; D006046:Gold
keywords: DNA; Diagnosis; Gold nanoparticles; Leishmania; Nanotechnology