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Multiplexed identification of different fish species by detection of parvalbumin, a common fish allergen gene: a DNA application of multi-analyte profiling (xMAP™) technology

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Abstract

Fish are a common cause of allergic reactions associated with food consumption, with parvalbumin being the major allergenic protein. Some fish-hypersensitive patients tolerate some fish species while being allergic to others. Reliable detection methods for allergenic fish species in foods are necessary to ensure compliance with food allergen labeling guidelines to protect fish-allergic consumers. The objective of this project was to develop a multi-analyte detection method for the presence of fish in food. Therefore, conserved parvalbumin exon sequences were utilized for the design of universal PCR primers amplifying intron DNA and small regions of exons flanking the enclosed intron from even very distantly related fish species. An assay for the identification of eight fish species was developed using xMAP™ technology with probes targeting species-specific parvalbumin intron regions. Additionally, a universal fish probe was designed targeting a highly conserved exon region located between the intron and the reverse primer region. The universal fish assay showed no cross-reactivity with other species, such as beef, pork, lamb, chicken, turkey, and shrimp. Importantly, with the exception of one notable case with fish in the same subfamily, species-specific detection showed no cross-reactivity with other fish species. Limits of detection for these eight species were experimentally estimated to range from 0.01% to 0.04%, with potential to increase the detection sensitivity. This report introduces a newly developed method for the multiplex identification of at least eight allergenic fish species in food, which could conceivably be extended to detect up to 100 species simultaneously in one sample.

Schematic overview of the xMAP™ assay. Amine-modified capture oligonucleotides were covalently coupled to color-coded MagPlex™-C magnetic carboxylated microspheres (Luminex Corporation, TX, USA) using a carbodiimide coupling method described by Fulton et al. [25]. Parvalbuminencoding DNA was amplified with real-time PCR, whereas one of the primers was labeled with biotin. Biotin-labeled DNA strands were hybridized to their corresponding capture oligonucleotides. The fluorophor streptavidin–phycoerythrin (SAPE) was added and bound to the biotin molecules. Reaction tubes containing the magnetic microspheres were put on a magnet, unbound oligonucleotides and SAPE was removed by washing. The reaction mixture was analyzed on the BioPlex 200 analyzer (BioRad, CA, USA). Microspheres were interrogated individually in a rapidly flowing fluid stream as they passed by two separate lasers. A red diode laser excited two fluorochromes contained within the colorcoded microspheres and a green laser excited the reporter fluorochrome (SAPE) bound to the microsphere surface. High-speed digital signal processing classified the microsphere based on its spectral address and quantified the reaction on the surface

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Acknowledgments

Gratitude is expressed to Jonathan Deeds, Ph.D. (FDA) for providing the authenticated fish samples. Appreciation is also expressed to Eric A. E. Garber, Ph.D. (FDA) and Steven R. Monday, Ph.D. (FDA) for guidance and critical reading of this work.

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  1. U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Regulatory Science, Division of Bioanalytical Chemistry, 5100 Paint Branch Parkway, College Park, MD, 20740, USA

    Sabine Hildebrandt

  2. FDA, CFSAN, HFS-716, 5100 Paint Branch Parkway, College Park, MD, 20740, USA

    Sabine Hildebrandt

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  1. Sabine Hildebrandt
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Correspondence to Sabine Hildebrandt.

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Hildebrandt, S. Multiplexed identification of different fish species by detection of parvalbumin, a common fish allergen gene: a DNA application of multi-analyte profiling (xMAP™) technology. Anal Bioanal Chem 397, 1787–1796 (2010). https://doi.org/10.1007/s00216-010-3760-2

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  • DOI: https://doi.org/10.1007/s00216-010-3760-2

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