Food Analytical Methods

, Volume 12, Issue 3, pp 811–825 | Cite as

Detection of Gluten-Rich Cereals in Processed Foods with Enhanced Sensitivity by Targeting Mitochondrial DNA Using PCR

  • Nahed Ahmed
  • Menghsiao MengEmail author


Since the correct labeling of foods is the only effective way of protecting celiac disease patients and people allergic to gluten, it is essential to have reliable methods to detect the presence of gluten-containing cereals in foods. DNA-based methods have their merits as complementary approaches to immunochemical assays to detect the possible gluten contamination in processed foodstuff. Nevertheless, insufficient sensitivity is a major drawback for DNA-based analytical methods. Accordingly, DNA markers within mitochondrial DNA were identified in this study as the targets of PCR for the detection of gluten-containing cereals. Primer pairs Mt1F/Mt1R and Mt2F/Mt2R target a unique DNA segment conserved among wheat, barley, rye, and oat, whereas Mt3F/Mt3R targets the other segment that is present only in wheat, barley, and rye. In regard to detection limits, all the three designed primers could detect the presence of 0.2 pg wheat, barley, and rye DNA, 10−5 (10 ppm) of wheat, barley, and rye DNA diluted with soya DNA, or 10−5 (10 ppm) of wheat flour mixed within corn flour. The extremely high copy numbers of mitochondrial genome per cell may explain and justify the astonishing sensitivity of the methods described in this study. The detection of wheat DNA in thermally processed foods by this method is evidence of the suitability and applicability of the method to examine foods with gluten-free labels.


Celiac disease Gluten-free foods Wheat allergy Gliadin PCR Molecular diagnosis 



We would like to thank Professor Weiming Leu at the Graduate Institute of Biotechnology, National Chung Hsing University, Taiwan, for her assistance with bioinformatics analysis. This study was financially supported by the Minister of Science and Technology, R.O.C. (Taiwan), under the grant MOST 106-2313-B-005-036-MY3.

Compliance with Ethical Standards

Conflict of Interest

Nahed Ahmed declares that he has no conflict of interest. Menghsiao Meng declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable.


  1. Allmann M, Candrian U, Höfelein C, Lüthy J (1993) Polymerase chain reaction (PCR): a possible alternative to immunochemical methods assuring safety and quality of food detection of wheat contamination in non-wheat food products. Z Lebensm Unters Forsch 196:248–251CrossRefPubMedGoogle Scholar
  2. Anonymous (2005) International Standard EN ISO 21571Google Scholar
  3. Arentz-Hansen H, Fleckenstein B, Molberg Ø, Scott H, Koning F, Jung G, Roepstorff P, Lundin KE, Sollid LM (2004) The molecular basis for oat intolerance in patients with celiac disease. PLoS Med 1(1):e1. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Blands J, Diamant B, Kallos P, Kallos-Deffner L, Lowenstein H (1976) Flour allergy in bakers. I Identification of allergenic fractions in flour and comparison of diagnostic methods. Int Arch Allergy Appl Immunol 52:392–406CrossRefPubMedGoogle Scholar
  5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  6. Catassi C, Fabiani E, Iacono G, D'Agate C, Francavilla R, Biagi F, Volta U, Accomando S, Picarelli A, de Vitis I, Pianelli G, Gesuita R, Carle F, Mandolesi A, Bearzi I, Fasano A (2007) A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease. Am J Clin Nutr 85:160–166CrossRefPubMedGoogle Scholar
  7. Ciclitira PJ, Moodie SJ (2003) Coeliac disease. Best Pract Res Cl Ga 17:181–195CrossRefGoogle Scholar
  8. Dahinden I, von Büren M, Lüthy J (2001) A quantitative competitive PCR system to detect contamination of wheat, barley or rye in gluten-free food for coeliac patients. Eur Food Res Tech 212:228–233CrossRefGoogle Scholar
  9. Debnath J, Martin A, Gowda LR (2009) A polymerase chain reaction directed to detect wheat glutenin: implications for gluten-free labelling. Food Res Int 42:782–787CrossRefGoogle Scholar
  10. Denham JM, Hill ID (2013) Celiac disease and autoimmunity: review and controversies. Curr Allergy Asthma Rep 13:347–353CrossRefPubMedPubMedCentralGoogle Scholar
  11. Haraszi R, Chassaigne H, Maquet A, Ulberth F (2011) Analytical methods for detection of gluten in food-method developments in support of food labeling legislation. J AOAC Int 94:1006–1025PubMedGoogle Scholar
  12. Kilmartin C, Lynch S, Abuzakouk M, Wieser H, Feighery C (2003) Avenin fails to induce a Th1 response in coeliac tissue following in vitro culture. Gut 52:47–52CrossRefPubMedPubMedCentralGoogle Scholar
  13. Köppel E, Stadler M, Lüthy J, HuÈbner P (1998) Detection of wheat contamination in oats by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Zeitschrift für Lebensmitteluntersuchung und-Forschung A 206:399–403CrossRefGoogle Scholar
  14. Mafra I, Ferreira IM, Oliveira MBP (2008) Food authentication by PCR-based methods. Eur Food Res Tech 227:649–665CrossRefGoogle Scholar
  15. Martín-Fernández B, Costa J, Oliveira MBP, López-Ruiz B, Mafra I (2015) Screening new gene markers for gluten detection in foods. Food Control 56:57–63CrossRefGoogle Scholar
  16. Mazzara M, Savini C, Delobel C, Broll H, Damant A, Paoletti C, van den Eede G (2008) Definition of minimum performance requirements for analytical methods of GMO Testing European Network of GMO Laboratories (ENGL). Joint Research Centre and Office for Official Publications of the European Communities. 8 p. OPOCE, BrusselsGoogle Scholar
  17. Mujico JR, Lombardía M, Mena MC, Méndez E, Albar JP (2011) A highly sensitive real-time PCR system for quantification of wheat contamination in gluten-free food for celiac patients. Food Chem 128:795–801CrossRefGoogle Scholar
  18. Ogihara Y, Yamazaki Y, Murai K, Kanno A, Terachi T, Shiina T, Miyashita N, Nasuda S, Nakamura C, Mori N, Takumi S, Murata M, Futo S, Tsunewaki K (2005) Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome. Nucleic Acids Res 33:6235–6250CrossRefPubMedPubMedCentralGoogle Scholar
  19. Olexova L, Dovičovičová L, Švec M, Siekel P, Kuchta T (2006) Detection of gluten-containing cereals in flours and “gluten-free” bakery products by polymerase chain reaction. Food Control 17:234–237CrossRefGoogle Scholar
  20. Piknová Ľ, Brežná B, Kuchta T (2008) Detection of gluten-containing cereals in food by 5′-nuclease real-time polymerase chain reaction. J Food Nutr Res 47:114–119Google Scholar
  21. Rooney JP, Ryde IT, Sanders LH et al (2015) PCR based determination of mitochondrial DNA copy number in multiple species. Methods Mol Biol 1241:23–38CrossRefPubMedPubMedCentralGoogle Scholar
  22. Sandberg M, Lundberg L, Ferm M, Yman I (2003) Real time PCR for the detection and discrimination of cereal contamination in gluten free foods. Eur Food Res Technol 217:344–349CrossRefGoogle Scholar
  23. Taylor SL (1987) Allergic and sensitivity reactions to food components. In: Hathcock JN (ed) Nutritional toxicology, vol II. Academic, New York, pp 173–197CrossRefGoogle Scholar
  24. Yamakawa H, Akiyama H, Endo Y et al (2007) Specific detection of wheat residues in processed foods by polymerase chain reaction. Biosci Biotechnol Biochem 71:2561–2564CrossRefPubMedGoogle Scholar
  25. 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 228:321–330CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.International Master Program of AgricultureNational Chung Hsing UniversityTaichungTaiwan
  2. 2.Graduate Institute of BiotechnologyNational Chung Hsing UniversityTaichungTaiwan

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