Kalinin Egor V, Chalenko Yaroslava M, Kezimana Parfait, Stanishevskyi Yaroslav M, Ermolaeva Svetlana A
Laboratory of Ecology of Pathogenic Bacteria, Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia; Institutue of Biochemical Technology and Nanotechnology, Peoples' Friendship University of Russia (RUDN University), 117198 Moscow, Russia.
Laboratory of Ecology of Pathogenic Bacteria, Gamaleya Research Center of Epidemiology and Microbiology, 123098 Moscow, Russia.
J Dairy Sci. 2023 Mar;106(3):1638-1649. doi: 10.3168/jds.2022-21997. Epub 2023 Jan 27.
The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.
革兰氏阳性菌单核细胞增生李斯特菌是一种污染乳制品的重要食源性病原体。与单核细胞增生李斯特菌密切相关的腐生李斯特菌属也是食品尤其是乳制品的常见污染物。为了区分单核细胞增生李斯特菌与非致病性李斯特菌属及其他细菌，开发了一种斑点免疫测定法。该免疫测定法基于针对表面毒力相关的单核细胞增生李斯特菌特异性InlB蛋白分泌形式的多克隆抗体。为了增加InlB的产量，细菌在添加了0.2%活性炭的脑心浸液琼脂（BHIC琼脂）上培养。将人工污染的生乳样品直接接种在BHIC琼脂上，然后进行斑点免疫测定，能够快速鉴定出浓度低至10 cfu/mL的单核细胞增生李斯特菌。使用所开发的方法，在14小时内获得初步结果，26小时后获得最终结果。对属于不同克隆复合体和系统发育谱系的单核细胞增生李斯特菌菌株、李斯特菌属及其他细菌物种进行了斑点免疫测定测试。结果表明，所开发的斑点免疫测定法在快速鉴定单核细胞增生李斯特菌方面具有卓越的特异性。
