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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

Abstract

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.

Zusammenfassung

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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Acknowledgments

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). https://doi.org/10.1007/s00003-011-0669-4

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  • DOI: https://doi.org/10.1007/s00003-011-0669-4

Keywords

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