European Food Research and Technology

, Volume 245, Issue 3, pp 559–567 | Cite as

Multiplex real-time PCR for the detection of insect DNA and determination of contents of Tenebrio molitor, Locusta migratoria and Achaeta domestica in food

  • René KöppelEmail author
  • Rafael Schum
  • Michael Habermacher
  • Cindy Sester
  • Lucia Eugeni Piller
  • Stefanie Meissner
  • Klaus Pietsch
Original paper


In Europe, edible insects are not a part of a common diet. But according to the Food and Agriculture Organization of the united nations (FAO), animal protein from insects could be an ecological, economic and healthy addition to human diets. In the EU there are currently no regulations on insects as food for human consumption. The production and sales of products containing insects are not prohibited. Several companies are producing and marketing food products containing insects already. In Switzerland, the recently revised food law allows and regulates insect production and products for human consumption. Like other ingredients, such food additives, insects have to be labeled correctly. To enforce such labeling, food control laboratories need analytical tools to detect insect ingredients and determine their species and quantity. We, therefore, developed a multiplex real-time PCR method detecting insect DNA generally and the three approved species specifically. The performance of this multiplex real-time PCR method was assessed during validation including data from other laboratories. These results indicate that the method is fit for purpose.


Multiplex real-time quantitative PCR Insect Tenebrio molitor Locusta migratoria Achaeta domestica 



We thank the Food Control Authority of the Canton Zürich for providing the resources for this work.

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 mammalian subjects.


  1. 1.
    Kambhampati S, Smith PT (1995) PCR primers for the amplification of four insect mitochondrial gene fragments. Insect Mol Biol 4(4):233–236CrossRefGoogle Scholar
  2. 2.
    Ji Y-J, Zhang D-X, He L-J (2003) Evolutionary conservation and versatility of a new set of primers for amplifying the ribosomal internal transcribed spacer regions in insects and other invertebrates. Mol Ecol Notes 3:581–585CrossRefGoogle Scholar
  3. 3.
    Pons J, Barraclough TG, Gomez-Zurita J, Cardoso A, Duran DP, Hazell S, Vogler AP (2006) Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Syst Biol 55(4):595–609CrossRefGoogle Scholar
  4. 4.
    Cognato AI (2006) Standard percent DNA sequence difference for insects does not predict species boundaries. J Econ Entomol 99(4):1037–1045CrossRefGoogle Scholar
  5. 5.
    Caterino MS, Cho S, Sperling FAH (2000) The current state of insect molecular systematics: a thriving tower of babel. Annu Rev Entomol 45:1–54CrossRefGoogle Scholar
  6. 6.
    Hebert PD, Cywinska A, Ball SL (2003) Biological identifications through DNA barcodes. Proc R Soc Lond B Biol Sci, 270(1512), 313–321CrossRefGoogle Scholar
  7. 7.
    Hebert PD, Gregory TR (2005) The promise of DNA barcoding for taxonomy. Syst Biol 54(5):852–859CrossRefGoogle Scholar
  8. 8.
    Park DS, Suh SJ, Hebert PDN, Oh HW, Hong KJ (2011) DNA barcodes for two scale insect families, mealybugs (Hemiptera: Pseudococcidae) and armored scales (Hemiptera: Diaspididae). Bull Entomol Res 101:429–434CrossRefGoogle Scholar
  9. 9.
    Van Hiel MB, Van Wielendaele P, Temmerman L, Van Soest S, Vuerinckx K, Huybrechts R, Simonet G (2009) Identification and validation of housekeeping genes in brains of the desert locust Schistocerca gregaria under different developmental conditions. BMC Mol Biol 10(1):56CrossRefGoogle Scholar
  10. 10.
    Xue JL, Salem TZ, Turney CM, Cheng XW (2010) Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses. J Virol Methods 163(2):210–215CrossRefGoogle Scholar
  11. 11.
    Majerowicz D, Alves-Bezerra M, Logullo R, Fonseca-de-Souza AL, Meyer-Fernandes JR, Braz GRC, Gondim KC (2011) Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae). Insect Mol Biol 20(6):713–722CrossRefGoogle Scholar
  12. 12.
    Teng X, Zhang Z, He G, Yang L, Li F (2012) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in four lepidopteran insects. J Insect Sci 12(60):1–17CrossRefGoogle Scholar
  13. 13.
    Reim T, Thamm M, Rolke D, Blenau W, Scheiner R (2013) Suitability of three common reference genes for quantitative real-time PCR in honey bees. Apidologie 44(3):342–350CrossRefGoogle Scholar
  14. 14.
    Yang C, Pan H, Liu Y, Zhou X (2014) Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris)(Hemiptera, Aphidiae). PloS One, 9(11), e110454CrossRefGoogle Scholar
  15. 15.
    Zhou X, Li Y, Liu S, Yang Q, Su X, Zhou L, Huang Q (2013) Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification. Gigascience 2(1):4CrossRefGoogle Scholar
  16. 16.
    Morf NV, Wood KL, Köppel R, Felderer N, Daniels M, Tenger B, Kratzer A (2013) A multiplex PCR method to identify bushmeat species in wildlife; forensics Forensic Sci Int Genet Suppl Ser, 4, 1, e202–e203CrossRefGoogle Scholar
  17. 17.
    Herbert PD, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes Proc Biol Sci 270 (1512) 313–321CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • René Köppel
    • 1
    Email author
  • Rafael Schum
    • 1
  • Michael Habermacher
    • 1
  • Cindy Sester
    • 2
  • Lucia Eugeni Piller
    • 2
  • Stefanie Meissner
    • 3
  • Klaus Pietsch
    • 3
  1. 1.Food Control Authority of the Canton ZürichZürichSwitzerland
  2. 2.Food Control Authority of the Canton NeuchâtelNeuchâtelSwitzerland
  3. 3.State Institute of Chemical and Veterinarian Analysis Freiburg BrFreiburgGermany

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