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Development of sensitive and specific real-time PCR systems for the detection of crustaceans in food

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Abstract

Crustaceans are known allergens for a remarkable number of people. For the detection of traces of crustaceans in food, a specific and sensitive real-time PCR method was developed. An approximately 205 bp long fragment of the mitochondrial 16S rRNA gene was chosen as molecular target region for the detection systems. The DNA sequence of this fragment was determined from 13 species belonging to different families and checked for homologies. Based on these data, primer–probe systems were developed for the economically relevant decapods within the class of Malacostraca belonging to the families Penaeidae, Palinuroidea, Astacoidea, Nephropoidea, Cancridae and Caridea. The specificity of the primer–probe systems was checked for inclusivity using DNA extracted from 17 different crustaceans. Exclusivity tests were carried out by analysing DNA samples derived from 21 mammals, six birds, 13 fishes, two molluscs and two insect species. Except for two systems, the molecular detection systems were optimised to be highly specific for the crustaceans. False positive signal was produced by DNA extracted from the hoverfly (Psilota rubra) in the system targeting the family Astacoidea and the common green lacewing (Chrysoperla carnea) in the systems targeting the family Astacoidea and Cancroidea. The LOD95% was close to the theoretical value of 2.96 copies per reaction. The sensitivity of the real-time PCR systems was determined using dilution series of crustacean DNA in rainbow trout DNA as animal matrix, and by artificial contamination of fish sticks and by artificial contamination of cassava chips with crustacean meat. The sensitivity ranging from 10 to 0.01 ppm is considered being appropriate for food analysis.

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Abbreviations

rRNA:

Ribosomal RNA

PCR:

Polymerase chain reaction

FAM:

6-Carboxyfluorescein

BBQ:

Black Berry Quencher

References

  1. Wüthrich B, Jäger L (2002) Nahrungsmittelallergien und-intoleranzen, Urban & Fischer München, 2. Auflage

  2. Alonso E, Zavala B, Escoda S (2006) Mantis Shrimp Allergy. Clinical Immunologie 16:394–396

    CAS  Google Scholar 

  3. EFSA (2006) Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the evaluation of mollusks for labelling purposes. EFSA J 327:1–25

    Google Scholar 

  4. Paschke A (2008) Proteine als Lebensmittelallergene. Nachr Chem 56:1005–1009

    Article  CAS  Google Scholar 

  5. United States Public Law (2004) Food Allergen Labeling and Consumer Protection Act of 2004. Public Law 108:282, 118, Stat. 905

  6. Regulation (EU) No 1169/2011of the European Parliament and of the Council of 25 October 2011 on the revision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004

  7. Rehbein H (2001) Identification of shrimp species by protein- and dna-based analytical methods. Annales Societatis Scientiarum Faeroensis Supplementum 28:195–205

    Google Scholar 

  8. Fuller HR, Goodwin PR, Morris GE (2006) An enzyme-linked immunosorbent assay (ELISA) for the major crustacean allergen, tropomyosin in food. Food Agric Immunol 17:43–52

    Article  CAS  Google Scholar 

  9. Seiki K, Oda H, Yoshioka H, Sakai S, Urisu A, Akiyama H, Ohno Y (2007) A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods. J Agric Food Chem 55:9345–9350

    Article  CAS  Google Scholar 

  10. Werner MT, Fæste CK, Egaas E (2007) Quantitative sandwich ELISA for the determination of tropomyosin from crustaceans in foods. J Agric Food Chem 55:8025–8032

    Article  CAS  Google Scholar 

  11. Brzezinski JL (2005) Detection of crustacean DNA and species identification using a PCR-restriction fragment length polymorphism method. J Food Prot 68:1866–1873

    Article  CAS  Google Scholar 

  12. Unterberger C, Luber F, Demmel A, Grünwald K, Huber I, Engel K-H, Busch U (2014) Simultaneous detection of allergenic fish, cephalopods and shellfish in food by multiplex ligation-dependent probe amplification. Eur Food Res Technol 239:559–566

    Article  CAS  Google Scholar 

  13. Cao J, Yu B, Ma L, Zheng Q, Zhao X, Xu J (2011) Detection of shrimp-derived components in food by real-time fluorescent PCR. J Food Protect 74:1776–1781

    Article  CAS  Google Scholar 

  14. Wolf C, Burgener M, Hübner P, Lüthy J (2000) PCR-RFLP analysis of mitochondrial DNA: differentiation of fish species. Lebensm Wiss Technol 33:144–150

    Article  CAS  Google Scholar 

  15. Brežná B, Hudecová L, Kuchta T (2006) A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food. Eur Food Res Technol 223:373–377

    Article  Google Scholar 

  16. Hupfer Ch, Waiblinger HU, Busch U (2007) Development and validation of a real-time PCR detection method for celery in food. Eur Food Res Technol 225:329–335

    Article  CAS  Google Scholar 

  17. Zeltner D, Glomb MA, Maede D (2009) Real-time PCR systems for the detection of the gluten-containing cereals wheat, spelt, kamut, rye, barley and oat. Eur Food Res Technol 228:321–330

    Article  CAS  Google Scholar 

  18. Köppel R, Dvorak V, Zimmerli F, Breitenmoser A, Eugster A, Waiblinger HU (2010) Two tetraplex real-time PCR for the detection and quantification of DNA from eight allergens in food. Eur Food Res Technol 230:367–374

    Article  Google Scholar 

  19. Tetzlaff C, Mäde D (2016) Development of a real-time PCR system for the detection of the potential allergen fish in food. Eur Food Res Technol. doi:10.1007/s00217-016-2799-5

    Google Scholar 

  20. Eischeid A, Kim BH, Kasko SM (2012) Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens. J Agric Food Chem 61(24):5669–5674. doi:10.1021/jf3031524

    Article  Google Scholar 

  21. Herrero B, Vieites JM, Espineira M (2012) Fast real-time PCR for the detection of crustacean allergen in foods. J Agric Food Chem 60:1893–1897

    Article  CAS  Google Scholar 

  22. International Organization for Standardization (2005) Microbiology of food and animal feeding stuffs—Polymerase chain reaction (PCR) for the detection of food-borne pathogens—general requirements and definitions. International Standard ISO 22174:2005. Geneva, Switzerland

  23. Rogers SO, Bendich AJ (1988) Extraction of DNA from plant tissues. Plant Mol Biol Man A6:1–10

    Google Scholar 

  24. International Organization for Standardization (2005) Foodstuffs—methods of analysis for the detection of genetically modified organisms and derived products—nucleic acid extraction. International Standard ISO 21571:2005. Geneva, Switzerland

  25. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DL (1997) Nucl Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  26. National Center for Biotechnology Information, U.S. National Library of Medicine. http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide. Accessed on 25 Jan 2017

  27. The European Bioinformatics Institute. Multiple sequence alignment. http://www.ebi.ac.uk/Tools/clustalw2/. New version: http://www.ebi.ac.uk/Tools/msa/clustalo/. Accessed on 25 Jan 2017

  28. Uhlig S, Frost K, Colson B, Simon K, Mäde D, Reiting R, Gowik P, Grohmann L (2015) Validation of qualitative PCR methods on the basis of mathematical-statistical modelling of the probability of detection. Accred Qual Assur 20:75–83

    Article  Google Scholar 

  29. Ballmer-Weber BK, Fernandez-Rivas M, Beyer K, Defernez M, Sperrin M, Mackie AR, Salt LJ, Hourihane JO, Asero R, Belohlavkova S, Kowalski M, de Blay F, Papadopoulos NG, Clausen M, Knulst AC, Roberts G, Popov T, Sprikkelman AB, Dubakiene R, Vieths S, van Ree R, Crevel R, Mills ENC (2015) How much is too much? Threshold dose distributions for 5 food allergens. J Allergy Clin Immunol 135:964–971

    Article  CAS  Google Scholar 

  30. Nordlee JA, Remington BC, Ballmer-Weber BK, Lehrer SB, Baumert JL, Taylor SL (2013) Threshold dose for shrimp: a risk characterization based on objective reactions in clinical studies. J Allergy Clin Immunol 131:AB88

    Article  Google Scholar 

  31. Taylor SL, Baumert JL, Kruizinga AG, Remington BC, Crevel RWR, Brooke-Taylor S, Allen KJ, Houben G (2014) Establishment of reference doses for residues of allergenic foods: report of the VITAL Expert Panel. Food Chem Toxicol 63:9–17

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are very grateful to Christiane Klemm and Katja Trübner-Mäde for their excellent technical assistance. We would like to thank John Church for carefully reading the manuscript and for helpful comments.

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  1. Landesamt für Verbraucherschutz Sachsen-Anhalt, Fachbereich Lebensmittelsicherheit, Freiimfelder Str. 68, 06112, Halle (Saale), Germany

    Dietrich Mäde & Diane Rohmberger

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  1. Dietrich Mäde
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  2. Diane Rohmberger
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Correspondence to Dietrich Mäde.

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All institutional and national guidelines for the care and use of animals were followed.

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Mäde, D., Rohmberger, D. Development of sensitive and specific real-time PCR systems for the detection of crustaceans in food. Eur Food Res Technol 243, 2105–2113 (2017). https://doi.org/10.1007/s00217-017-2911-5

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  • DOI: https://doi.org/10.1007/s00217-017-2911-5

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