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Detection of honeybee bacterial pathogens with upconversion-linked immunosorbent assay

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PASTUCHA Matěj ODSTRČILÍKOVÁ Eliška POLÁCHOVÁ Veronika BRANDMEIER Julian HLAVÁČEK Antonín GORRIS Hans-Heiner FARKA Zdeněk SKLÁDAL Petr

Rok publikování 2021
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
Popis European and American Foulbroods (EFB and AFB) are the most serious honeybee diseases caused by bacteria. In the case of an outbreak of either disease, an effective diagnosis method is necessary, sensitive enough to detect the pathogen before the manifestation of clinical symptoms. Bacterial pathogens are traditionally detected by cultivation on selective media. Cultivations are lengthy but very sensitive and have low requirements on the laboratory equipment. However, their limits become apparent when the bacterial strain is difficult to cultivate or when the results are needed quickly. Enzyme-linked immunosorbent assay (ELISA) is a classic method for the detection of various analytes. Modern nanoparticle labels provide improved sensitivity and enhanced properties compared to conventional labels. Luminescent nanoparticles can substitute traditional fluorophores and particularly the photon-upconversion nanoparticles (UCNP) bring the intriguing possibility of background-free luminescence measurement. We developed novel polyclonal antibodies detecting Melissococcus plutonius (EFB) and Paenibacillus larvae (AFB). In both cases, a “traditional” ELISA was not sensitive enough to reveal the early stages of the diseases, and we improved it by applying streptavidin-labeled UCNPs (BSA-coated UCNPs for EFB and PEGylated for AFB). With the upconversion readout, we achieved an LOD of 3.4×10^2 CFU mL-1 for M. plutonius and 2.9×10^3 CFU/mL for P. larvae. This represents a 400- and 22-fold improvement over the ELISA. Furthermore, we have successfully detected bacteria in spiked samples of bees, larvae, and hive debris.
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