Development of Fluorescence-Based Liposome Immunoassay for Detection of Cronobacter muytjensii in Pure Culture

Cronobacter spp. are important foodborne pathogens that carry a very high risk of infection to neonates as well as immunocompromised individuals. In the present study, fluorescence-based liposome immunoassay was developed as a new sensitive and rapid diagnostic system for detection of Cronobacter muytjensii (C. muytjensii). Liposomes (size, 206 nm) used in this study were made from cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], and sulforhodamine B (SRB). The outer surface of liposome was conjugated with rabbit anti-C. muytjensii IgG in order to develop immunoliposome. The immunoliposome was incubated with C. muytjensii, which was coated on a 96-well plate. Immunoliposomes bound to C. muytjensii were lysed with 30 mM octyl beta-d-glucopyranoside, after which the SRB fluorescence signal was measured at an excitation wavelength of 550 nm and emission wavelength of 585 nm. The signal was directly proportional to the amount of bacterial cells in the test sample. The developed fluorescence-based liposome immunoassay was confirmed to be highly specific to C. muytjensii with a detection limit of 6.3 x 10(4) CFU ml(-1) in pure culture as well as sensitive, efficient, and rapid when compared to culture-based methods. Based on its rapid efficiency and low cost, this fluorescence-based liposome immunoassay may be used to develop diagnostic kits for C. muytjensii detection.