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

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Abstract

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.

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Acknowledgements

The authors would like to thank Ted Knoy for his editorial assistance.

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Authors and Affiliations

  1. Department of Food Science and Biotechnology, National Chung Hsing University, 145, Xingda Rd., Taichung, 402, Taiwan

    Hsin-Yi Yin & Hsiao-Wei Wen

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  1. Hsin-Yi Yin
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  2. Hsiao-Wei Wen
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Correspondence to Hsiao-Wei Wen.

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Funding

This study was funded by National Science Council of the Republic of China, Taiwan (No. NSC 99-2627-M-005-002).

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This article does not contain any studies with human or animal subjects.

Conflict of Interest

Hsin-Yi Yin declares that he has no conflict of interest. Hsiao-Wei Wen declares that he has no conflict of interest.

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Not applicable.

Electronic Supplementary Material

ESM 1

Optimization of amount of antibody coated on protein G magnetic beads. Different concentrations of anti-digoxigenin (DIG) antibodies (0–250 μg mL-1) were mixed with ‬protein G magnetic beads for 30 min at 37 oC to produce immunomagnetic beads (IMBs), which were used in capturing DIG and biotin dual-labeled amplicons. Bound amplicons were measured using streptavidin conjugated horseradish peroxidase (SA-HRP). (DOCX 12 kb)

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Yin, HY., Wen, HW. Dual-Labeled PCR-Based Immunofluorescent Assay for the Rapid and Sensitive Detection of Enterotoxic Staphylococcus aureus Using Cocktail-Sized Liposomal Nanovesicles as Signal Enhancer. Food Anal. Methods 10, 3264–3274 (2017). https://doi.org/10.1007/s12161-017-0893-3

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