Dual-Labeled PCR-Based Immunofluorescent Assay for the Rapid and Sensitive Detection of Enterotoxic Staphylococcus aureus Using Cocktail-Sized Liposomal Nanovesicles as Signal Enhancer

• Published in: Food analytical methods 2017-10, Vol.10 (10), p.3264-3274
• Main Authors: Yin, Hsin-Yi, Wen, Hsiao-Wei
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Assaying; Bacteria; Beads; Biotin; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Digoxigenin; Dyes; E coli; Encapsulation; Enrichment; Fluorescence; Fluorescent dyes; Fluorescent indicators; Food contamination; Food contamination & poisoning; Food poisoning; Food Science; Lysis; Microbiology; Milk; Optimization; Pathogens; Poisoning; Polymerase chain reaction; Primers; Salmonella; Sea gene; Staphylococcal enterotoxin A
• ISSN: 1936-9751; 1936-976X
• DOI: 10.1007/s12161-017-0893-3

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 10 1  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.