dual-labeled pcr-based immunofluorescent assay for the rapid and sensitive detection of enterotoxic staphylococcus aureus using cocktail-sized liposomal nanovesicles as signal enhancer

Staphylococcus aureus is a major foodborne pathogen worldwide, and as little as 1 μg of staphylococcal enterotoxins (ses) can cause food poisoning. among ses, enterotoxin a is the most common toxin that causes staphylococcal food poisoning. hence, this work has developed a dual-labeled pcr-based immunofluorescent assay using anti-digoxigenin (dig) antibody-tagged immunomagnetic beads (imbs) as the capture reagent and cocktail-sized neutravidin-tagged liposomal nanovesicles (na-lns) that encapsulate fluorescent dyes as the detection reagent. in this approach, the amplicon of sea gene was doubly labeled with dig and biotin using modified primers and biotin-11-dutp. the system depends on the immunocapture of imb to pre-concentrate the labeled amplicons, which were further quantified using cocktail-sized na-lns, based on the release and subsequent measurement of encapsulated fluorescent dyes following the lysis of na-lns. after optimization, the developed assay could detect s. aureus and differentiate it from other common foodborne bacteria, such as salmonella enterica and escherichia coli, with a limit of detection (lod) of 101 cfu ml−1 without pre-enrichment. with a 2-h pre-enrichment, this developed assay could detect as little as 1 cfu in 25 ml of milk within a workday. hence, this work established a rapid and sensitive pcr-based immunofluorescent assay using liposomal nanovesicles as an instant signal enhancer to detect the contamination of enterotoxic s. aureus in milk.