Quantification of the allergen soy (Glycine max) in food using digital droplet PCR (ddPCR)
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To meet the increasing need for quantification of allergens and to have an alternative to commercially available ELISA and PCR systems, the Austrian Agency for Health and Food Safety started establishing in-house PCR systems. To obtain low limits of detection (LOD) and quantification (LOQ), target sequences are preferably sought in multicopy genomes like mitochondrial- or chloroplast DNA. These molecules are of high but varying abundance even among tissues of the same organism. Beyond that, DNA might be degraded by processes of food manufacturing which additionally affects their quantification. Therefore, a reliable correlation of the allergen portion in a sample and its chloroplast-DNA concentration cannot be preassumed. This incoherence is not further considered (e.g., by a matrix-related reference material), and therefore, our quantitative results can only be understood as the mass of soy which maintained its biochemical activity, related to the soy content of the reference material used. To convert absolute results expressed in copies per microliter (Cp/µL) as obtained by digital droplet PCR (ddPCR) into a unit of mass fraction (e.g., milligram per kilogram), a conversion function is generated by the measurement of a reference material in the same run. For the specific detection and quantification of the allergenic ingredient soy (Glycine max) in food a primer/probe system has been developed which amplifies a 140 bp product of the ndhH gene of the chloroplast DNA. It is specific for soy and does not react with even closely related plant species. Digital droplet PCR (ddPCR) was selected for quantification for its particular advantages and the method has been validated in-house. It was found to be applicable to various matrices including meat products, flour, milk, and fatty creams, with recovery rates between 60 and 100%. The limit of detection and the limit of quantification (LOQ) are 0.16 mg/kg and 0.60 mg/kg, respectively. Repeated analysis of analyte-free food matrices spiked with reference material provided acceptable values for precision: The relative standard deviation (RSDoverall) of the whole method (including DNA extraction) is below 25%. The recovery of pure soy material (pulverized beans) was between 112.5 and 135.0%. The presented method is shown to be reliable and accurate, provided that samples and reference material are extracted and amplified in the same way.
KeywordsChloroplast DNA Soybean Quantification Digital droplet PCR Food allergens
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Compliance with ethics requirements
This article does not contain any studies with human or animal subjects.
- 3.EU Regulation No 1169/2011 of the European Parliament and of the Council. Off J EU 304:18–63Google Scholar
- 10.Lebensmittel—Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren—Teil 5: Senf (Sinapis alba) sowie Soja (Glycine max)—Qualitativer Nachweis einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time PCR. CEN/TS 15634-5:2016Google Scholar
- 12.Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2004) Molekularbiologie der Zelle, 4th edn. Wiley-VCH, Weinheim, p 922fGoogle Scholar
- 13.Encyclopædia B (2017) https://www.britannica.com/science/chloroplast. Accessed 13 Feb 2018
- 14.Biologie-Schule Das Nachschlagewerk für Biologie (2018) http://www.biologie-schule.de/pflanzenzelle.php. Accessed 13 Feb 2018
- 15.National Center for Biotechnology Information (2016) https://www.ncbi.nlm.nih.gov/. Accessed 30 Jul 2016
- 17.Droplet, Digital™ PCR applications guide—Bio-Rad http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf. Accessed 13 Aug 2015
- 18.Bio-Rad Laboratories I ddPCR™ Supermix for Probes (No dUTP). Product Insert, Rev DGoogle Scholar
- 19.Dagata JA, Farkas N, Kramer J (2016) Method for measuring the volume of nominally 100 µm diameter spherical water-in-oil emulsion droplets. NIST Spec Publ 260(184):260–284Google Scholar
- 23.Žel J, Mazzara M, Savini C, Cordeil S, Camloh M, Stebih D, Cankar K, Gruden K, Morisset D, Van den Eede G (2008) Method validation and quality management in the flexible scope of accreditation: an example of laboratories testing for genetically modified organisms. Food Anal Methods. https://doi.org/10.1007/s12161-008-9016-5 Google Scholar
- 25.Grohmann L, Broll H, Dagand E, Hildebrandt S, Hübert P, Kiesecker H, Lieske K, Mäde D, Joachim Mankertz D, Ralf Reiting D, Manuela Schulze D, Speck B, Uhlig S, Wahler D, Waiblinger H-U, Woll K, Zur K (2016) Guidelines for the single-laboratory validation of qualitative real-time PCR methods. Bundesamt für Verbraucherschutz und Lebensmittelsicherheit (BVL), BraunschweigGoogle Scholar
- 26.Definition of minimum performance requirements for analytical methods of GMO testing (2015) European network of GMO laboratories (ENGL). http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm. Accessed 14 Apr 2016
- 27.AWI 20395 Biotechnology-guidelines for evaluating the performance of targeted nucleic acid quantification methods: Part 1 qPCR and dPCR (2017). ISO AWI 20395 (v4.3), vol unpublishedGoogle Scholar
- 28.Hougs L, Zel J, Charles-Delobel C, Burns M, Charels D, Ciabatti I (2011) Verification of analytical methods for GMO testing when implementing interlaboratory validated methods. In: ENGL working group on “Method Verification” of the joint research centre of the European Commission Luxembourg: JRC Scientific and Technical Reports Publications Office of the European UnionGoogle Scholar
- 31.Deprez L, Corbisier P, Kortekaas AM, Mazoua S, Beaz Hidalgo R, Trapmann S, Emons H (2016) Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material. Biomol Detect Quantif 9:29–39. https://doi.org/10.1016/j.bdq.2016.08.002 CrossRefGoogle Scholar