20.500.12556/RUL-175367 Campylobacter jejuni biofilm assessment by Nanoluc luciferase assay Campylobacter jejuni, a widespread pathogen found in birds and mammals, poses a significant risk for zoonosis worldwide despite its susceptibility to environmental and food-processing stressors. One of its main survival mechanisms is the formation of biofilms that can withstand various food-processing stressors, which is why efficient methods for assessing biofilms are crucial. Existing methods, including the classical culture-based plate counting method, biomass-staining methods (e.g., crystal violet and safranin), DNA-staining methods, those that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts and resazurin), immunofluorescence with flow cytometry or fluorescence microscopy, and PCR-based methods for quantification of bacterial DNA, are diverse but often lack specificity, sensitivity, and suitability. In response to these limitations, we propose an innovative approach using NanoLuc as a reporter protein. The established protocol involves growing biofilms in microtiter plates, washing unattached cells, adding Nano-Glo luciferase substrate, and measuring bioluminescence. The bacterial concentrations in the biofilms are calculated by linear regression based on the calibration curve generated with known cell concentrations. The NanoLuc protein offers a number of advantages, such as its small size, temperature stability, and highly efficient bioluminescence, enabling rapid, non-invasive, and comprehensive assessment of biofilms together with quantification of a wide range of cell states. Although this method is limited to laboratory use due to the involvement of genetically modified organisms, it provides valuable insights into C. jejuni biofilm dynamics that could indirectly help in the development of improved food safety measures. bioluminescence bioflm assay NanoLuc viable but nonculturable food safety Campylobacter jejuni bioluminiscenca biofilm luciferaza NanoLuc varnost živil true false true Angleški jezik Slovenski jezik Članek v reviji 2025-10-24 13:16:15 2025-10-24 13:16:19 2025-12-10 03:43:48 0000-00-00 00:00:00 2025 0 0 10 str. iss. 4, art. e5192 Vol. 15 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 579.2.088 2331-8325 10.21769/BioProtoc.5192 221752067 cukajne_Bio-protocol5192.pdf cukajne_Bio-protocol5192.pdf 1 5AE3E6DAF879FD7EC75B31EFAF8BAA4D 35b91760878b3819487f3f4caa458a5a5092491b377781125fa010683c383174 e1edf188-b0ca-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=220862 https://bio-protocol.org/en/bpdetail?id=5192&type=0 1 9534b7c9-b0ca-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=220860 Biotehniška fakulteta 0 0 0