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Evaluation of real-time PCR results at the limit of detection

Bewertung von real-time PCR-Befunden im Bereich der Nachweisgrenze


Real-time polymerase chain reaction (real-time PCR) facilitates to detect DNA fragments at very low copy numbers. Positive results, e.g. for unauthorized genetically modified organism (GMO) contamination or for allergens, may raise safety concerns and have far-reaching consequences. However, in case of very low concentrations of DNA samples, results for the same product lot or even for identical samples from different laboratories may differ. Therefore, an approach for a standardized interpretation and reporting of results obtained by real-time PCR at the limit of detection (LOD) is proposed. A quality control DNA sample containing the target at the LOD (95 %) is analysed in parallel with the real DNA sample and the respective C T values are compared. In addition, practical approaches for in house and precision-based estimation of the LOD are presented. The proposed approach may also contribute to the current discussion on implementing a technical solution to handle DNA traces in specimen, e.g. for the detection of unauthorized GMO.


Mittels real-time PCR ist ein Nachweis von DNS in sehr geringer Kopienzahl möglich. Positive Befunde, etwa bei der Analytik nicht zugelassener GVO oder von Lebensmittelallergenen können weitreichende Konsequenzen haben. Besonders wenn der nachzuweisende Stoff nur in sehr niedrigen Konzentrationen in einer Probe enthalten ist, kann dies bei der Untersuchung durch mehrere Laboratorien selbst bei identischen Proben zu unterschiedlichen Ergebnissen führen. Es wird daher ein Ansatz vorgeschlagen, wie Ergebnisse der real-time PCR im Bereich der Nachweisgrenze möglichst einheitlich interpretiert und berichtet werden können. Dazu wird Qualitätskontroll-DNS, welche die Zielsequenz im Bereich der Nachweisgrenze (LOD 95 %) enthält, parallel zur DNS aus der Probe untersucht und die CT-Werte miteinander verglichen. Zusätzlich wird ein praktisches Verfahren, beruhend auf validierten Daten, zur Abschätzung der Nachweis- bzw. Erfassungsgrenze beschrieben. Der vorgeschlagene Ansatz soll auch zur derzeitigen Diskussion bezüglich der Einführung einer technischen Lösung für den Umgang mit Spurenbefunden in Proben beitragen, zum Beispiel beim Nachweis nicht zugelassener GVO.

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  • AFNOR (2003) Association française de normalisation. Norme expérimentale AFNOR XP V03-020-2 (septembre 2003). Produits alimentaires—Détection et quantification des organismes végétaux génétiquement modifiés et produits derives. Partie 2: Méthodes quantitatives basées sur la réaction de polymérisation en chaîne

  • Burns M, Valdivia H (2008) Modelling the limit of detection in real-time quantitative PCR. Eur Food Res Technol 226:1513–1524

    Article  CAS  Google Scholar 

  • Burns M, Burrell A, Foy C (2010) The applicability of digital PCR for the assessment of detection limits in GMO analysis. Eur Food Res Technol 231:353–362

    Article  CAS  Google Scholar 

  • CGC (2010) Canadian Grain Commission. Sampling and testing protocol for Canadian flaxseed exported to the European Union.

  • Codex (2010) Codex Alimentarius Commission. Thirty-third session 5–9 July 2010. Proposed draft guidelines on Guideline on performance criteria and validation of methods for the detection, identification and quantification of specific DNA sequences and specific proteins in foods. ALINORM 10/33/23 Appendix III

  • DIN 32645 (2008) Chemische Analytik—Nachweis-, Erfassungs- und Bestimmungsgrenze unter Wiederholbedingungen—Begriffe, Verfahren, Auswertung. Beuth-Verlag, Berlin

  • EC (2003) Regulations (EC) No 1829/2003 and 1830/2003 of the European parliament and the council of 22.09.2003. Offic J L 268:1–29

    Google Scholar 

  • EC (2006) European Commission. Commission decision 2006/601/EC from 06.11.2006 on emergency measures regarding the non-authorised genetically modified organism LL RICE 601 in rice products. Offic J L 306:17–20

    Google Scholar 

  • EC (2009) European Commission. Joint Research Centre (2009). Report on the verification of the performance of a construct-specific assay for the detection of flax CDC Triffid Event FP 967 using real-time PCR. CRL-EM-01/09.

  • ENGL (2008) European Network of GMO Laboratories. Definition of minimum performance requirements for analytical methods of GMO testing.

  • GDCh (2005) German Food Chemist Society. WG “Biochemical and molecular biological analysis” (2005). Current questions in GMO-analysis (German language). Lebensmittelchemie 59:105–107.

    Google Scholar 

  • Grohmann L, Brünen-Nieweler C, Nemeth A, Waiblinger HU (2009) Collaborative trial validation studies of real-time PCR based screening methods for detection of the bar gene and the ctp2-cp4epsps construct. J Agric Food Chem. doi:10.1021/jf901598

  • Hübner P, Waiblinger HU, Pietsch K, Brodmann P (2001) Validation of PCR methods for quantitation of genetically modified plants. J AOAC Int 84:1855

    PubMed  Google Scholar 

  • ISO 24276 (2006) Foodstuffs—nucleic acid based methods of analysis for the detection of genetically modified organisms and derived products. General requirements. 05-2006

  • IUPAC (2006) Compendium of chemical terminology, 2nd edn (the “Gold Book”). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). XML on-line corrected version: (2006)

  • Lorenz R (1996) Basic principles of biometry. Gustav Fischer Verlag, Stuttgart (German language)

  • Reiting R, Broll H, Waiblinger HU, Grohmann L (2007) Collaborative study of a T-nos real-time PCR method for screening of genetically modified organisms in food products. J Verbraucherschutz Lebensmittelsicherheit 2:116–121

    Article  CAS  Google Scholar 

  • Valoo 2.3. Analytik Software. Leer, Germany

  • Waiblinger HU, Ernst B, Anderson A, Pietsch K (2007) Validation and collaboration study of a P35S and T-nos duplex real-time PCR screening method to detect genetically modified organisms in food products. Eur Food Res Technol 226:1221–1228

    Article  Google Scholar 

  • Working group “Genetical engineering” of the Federal States in Germany (2006) Concept for analysis of seeds for genetically modified plants.

Download references


We thank Dr. Thomas Holzhauser, Paul-Ehrlich Institut, Langen (Germany) for his helpful comments on the paper.

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Correspondence to Hans-Ulrich Waiblinger.

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Waiblinger, HU., Graf, N., Broll, H. et al. Evaluation of real-time PCR results at the limit of detection. J. Verbr. Lebensm. 6, 411–417 (2011).

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  • Real-time PCR
  • Limit of detection
  • GMO
  • Allergens
  • Zero tolerance