title: Detection of pathogenic mycobacteria based on functionalized quantum dots coupled with immunomagnetic separation.
abstract: Mycobacteria have always proven difficult to identify due to their low growth rate and fastidious nature. Therefore molecular biology and more recently nanotechnology, have been exploited from early on for the detection of these pathogens. Here we present the first stage of development of an assay incorporating cadmium selenide quantum dots (QDs) for the detection of mycobacterial surface antigens. The principle of the assay is the separation of bacterial cells using magnetic beads coupled with genus-specific polyclonal antibodies and monoclonal antibodies for heparin-binding hemagglutinin. These complexes are then tagged with anti-mouse biotinylated antibody and finally streptavidin-conjugated QDs which leads to the detection of a fluorescent signal. For the evaluation of performance, the method under study was applied on Mycobacterium bovis BCG and Mycobacterium tuberculosis (positive controls), as well as E. coli and Salmonella spp. that constituted the negative controls. The direct observation of the latter category of samples did not reveal fluorescence as opposed to the mycobacteria mentioned above. The minimum detection limit of the assay was defined to 10(4) bacteria/ml, which could be further decreased by a 1 log when fluorescence was measured with a spectrofluorometer. The method described here can be easily adjusted for any other protein target of either the pathogen or the host, and once fully developed it will be directly applicable on clinical samples.
authors.affiliation: Department of Animal Science and Aquaculture, Agricultural University of Athens, Athens, Greece.
authors.forename: Emmanouil
authors.initials: E
authors.lastname: Liandris
authors.forename: Maria
authors.initials: M
authors.lastname: Gazouli
authors.forename: Margarita
authors.initials: M
authors.lastname: Andreadou
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Sechi
authors.forename: Valentina
authors.initials: V
authors.lastname: Rosu
authors.forename: John
authors.initials: J
authors.lastname: Ikonomopoulos
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D004926:Escherichia coli / Q000166:cytology / Q000302:isolation & purification; D018189:Immunomagnetic Separation / Q000379:methods*; D057230:Limit of Detection; D008280:Magnetics; D051379:Mice; D008856:Microscopy, Fluorescence; D008863:Microspheres; D009161:Mycobacterium / Q000166:cytology / Q000276:immunology / Q000302:isolation & purification*; D009163:Mycobacterium bovis / Q000166:cytology / Q000302:isolation & purification; D045663:Quantum Dots
chemical_list: D000907:Antibodies, Bacterial