European Food Research and Technology

, Volume 227, Issue 6, pp 1699–1709 | Cite as

Detection of feed-derived maize DNA in goat milk and evaluation of the potential of horizontal transfer to bacteria

  • Aurora Rizzi
  • Lorenzo Brusetti
  • Stefania Arioli
  • Kaare M. Nielsen
  • Isabella Tamagnini
  • Alberto Tamburini
  • Claudia Sorlini
  • Daniele Daffonchio
Original Paper


The presence and the transforming capacity of feed-derived DNA in milk obtained from eight lactating goats fed on maize E176 silage were evaluated. The presence of single- and multi-copy maize genes was monitored by real-time PCR and conventional PCR. Chromosomal and plastid DNA extracted directly from maize flour and silage were readily amplifiable by conventional PCR, however, only chloroplast-specific gene fragments of 199 and 532 bp were detected in about 60 and 20%, respectively, of the milk samples analysed. Quantification by real time PCR yielded 9.5 (±6.7) × 102 plant gene copies/mL of milk sediment. In contrast, all milk samples were negative for the chromosomally located maize zein gene or the E176 specific cry1Ab transgene. The minimum concentration of plant DNA required for detection was 0.01 ng/mL raw milk for the chloroplast-specific fragment and 1 ng/mL for the cry1Ab transgene. The detection limit was determined by spiking milk samples with plant DNA prior to DNA extraction. The transformation capability of DNA in milk was evaluated after constructing a marker rescue system in Acinetobacter baylyi strain BD413 based on recombinational repair of the bla TEM gene. Two systems were developed that allowed the plant marker gene to recombine with the bacterial chromosome [A. baylyi BD413 (pUC-bla)] or plasmids [A. baylyi BD413 (pBBR1MCS-2Φ)]. The two systems showed the same efficiency of transformation, yielding 10−5 transformants per recipient cell (t/r) using plasmid pUC18 or a 1,873 bp fragment as donor DNA, and 3.5 × 10−11 t/r using DNA isolated from flour (E176). No transformants were detected when exposing the recipient bacterium to DNA extracted from maize (E176) silage or from milk obtained from goats feed maize (E176).


Genetically modified maize DNA detection Real time PCR Bacterial natural transformation Marker rescue Biosafety Risk assessment 



Genetically modified


Horizontal gene transfer


Polymerase chain reaction


Ultra high temperature



We are indebt to L.N. Ornston, R.G. Kok, and D.M. Young for providing the plasmid pZR80. We thank V. Garavaglia for the help with the experimental feeding design for the goats. This work was supported in part by a grant from ANPA (Agenzia Nazionale per la Protezione dell’Ambiente), contract “Sviluppo di metodologie per il monitoraggio di vegetali transgenici e derivati e della diffusione dei transgeni nell’ambiente”. K. M. Nielsen was supported by the Norwegian Research Council, and D. Daffonchio and K. M. Nielsen acknowledge support from the Fondazione Diritti Genetici, funded by the Cariplo Foundation, Italy.


  1. 1.
    Alexander TW, Reuter T, Aulrich K, Sharma R, Okine EK, Dixon WT, McAllister TA (2007) Anim Feed Sci Technol 133:31–62CrossRefGoogle Scholar
  2. 2.
    Andersson SGE, Zomorodipour A, Andersson JO, Sicheritz-Ponten T, Alsmark UCM, Podowski RM, Naslund AK, Eriksson A-S, Winkler HH, Kurland CG (1998) Nature 396:133–140CrossRefGoogle Scholar
  3. 3.
    Bauer F, Hertel C, Hammes WP (1999) System Appl Microbiol 22:161–168Google Scholar
  4. 4.
    Bensasson D, Boore JL, Nielsen KM (2004) Heredity 92:483–489CrossRefGoogle Scholar
  5. 5.
    Berdal KG, Holst-Jensen A (2001) Eur Food Res Technol 213:432–438CrossRefGoogle Scholar
  6. 6.
    Blomqvist T, Steinmoen H, Havarstein LS (2006) Appl Environ Microbiol 72:6751–6756CrossRefGoogle Scholar
  7. 7.
    Bräutigam M, Hertel C, Hammes WP (1997) FEMS Microbiol Lett 155:93–98CrossRefGoogle Scholar
  8. 8.
    Brusetti L, Rizzi A, Abruzzese A, Sacchi GA, Ragg E, Bazzicalupo M, Sorlini C, Daffonchio D (2008) Environ Biosafety Res 7:11–24CrossRefGoogle Scholar
  9. 9.
    Calsamiglia S, Hernandez B, Hartnell GF, Phipps R (2007) J Dairy Sci 90:4718–4723CrossRefGoogle Scholar
  10. 10.
    Chiter A, Forbes JM, Blair GE (2000) FEBS Lett 481:164–168CrossRefGoogle Scholar
  11. 11.
    De Vries J, Wackernagel W (1998) Mol Gen Genet 257:606–613CrossRefGoogle Scholar
  12. 12.
    De Vries J, Meier P, Wackernagel W (2001) FEMS Microbiol Lett 195:211–215CrossRefGoogle Scholar
  13. 13.
    Einspanier R, Klotz A, Kraft J, Aulrich K, Poser R, Schwägele F, Jahreis G, Flachowsky G (2001) Eur Food Res Technol 212:129–134CrossRefGoogle Scholar
  14. 14.
    Authority European Food Safety (2004) EFSA J 48:1–18Google Scholar
  15. 15.
    Gebhard F, Smalla K (1998) Appl Env Microbiol 64:1550–1554Google Scholar
  16. 16.
    Hupfer C, Hotzel H, Sachse K, Engel KH (1997) Z Lebensm Unters Forsch A 205:442–445CrossRefGoogle Scholar
  17. 17.
    Hupfer C, Mayer J, Hotzel H, Sachse K, Engel KH (1999) Eur Food Res Technol 209:301–304CrossRefGoogle Scholar
  18. 18.
    James C (2006) ISAAA briefs no. 35. ISAAA, Ithaca, NYGoogle Scholar
  19. 19.
    Juni E (1972) J Bacteriol 112:917–931Google Scholar
  20. 20.
    Kay E, Vogel TM, Bertolla F, Nalin R, Simonet P (2002) Appl Environm Microbiol 68:3345–3351CrossRefGoogle Scholar
  21. 21.
    Kay S, Van den Eede G (2001) Nat Biotechnol 19:405CrossRefGoogle Scholar
  22. 22.
    Kharazmi M, Hammes WP, Hertel C (2002) System Appl Microbiol 25:471–477CrossRefGoogle Scholar
  23. 23.
    Kharazmi M, Sczesny S, Blaut M, Hammes WP, Hertel C (2003) Appl Environ Microbiol 69:6121–6127CrossRefGoogle Scholar
  24. 24.
    Klotz A, Einspanier R (2001) Milchwissenschaft 56:67–70Google Scholar
  25. 25.
    Kok RG, Young DM, Ornston LN (1999) Appl Environ Microbiol 65:1675–1780Google Scholar
  26. 26.
    Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RMII, Peterson KM (1995) Gene 166:175–176CrossRefGoogle Scholar
  27. 27.
    Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV (1993) Bio-Technology 11:194–200Google Scholar
  28. 28.
    Lipkin E, Shalom A, Khatib H, Soller M, Friedmann A (1993) J Dairy Sci 76:2025–2032Google Scholar
  29. 29.
    Lorentz MG, Wackernagel W (1994) Microbiol Rev 58:563–602Google Scholar
  30. 30.
    Markmann-Mulisch U, Subramanian AR (1988) Eur J Biochem 170:507–514CrossRefGoogle Scholar
  31. 31.
    Nemeth A, Wurz A, Artim L, Charlton S, Dana G, Glenn K, Hunst P, Jennings J, Shilito R, Song P (2004) J Agric Food Chem 52:6129–6135CrossRefGoogle Scholar
  32. 32.
    Nielsen KM, van Weerelt DM, Berg TN, Bones AM, Hagler AN, Van Elsas JD (1997) Appl Environ Microbiol 63:1945–1952Google Scholar
  33. 33.
    Nielsen KM, Bone AM, Smalla K, Van Elsas JD (1998) FEMS Microbiol Rev 22:79–103Google Scholar
  34. 34.
    Nielsen KM, van Elsas JD, Smalla K (2000) Appl Environ Microbiol 66:1237–1242CrossRefGoogle Scholar
  35. 35.
    Nielsen KM, van Elsas JD (2001) Soil Biol Biochem 33:345–357CrossRefGoogle Scholar
  36. 36.
    Palmen R, Hellingwerf KJ (1997) Gene 192:179–190CrossRefGoogle Scholar
  37. 37.
    Phipps RH, Deaville ER, Maddison BC (2003) J Dairy Sci 86:4070–4078CrossRefGoogle Scholar
  38. 38.
    Phipps RH, Jones AK, Tingey AP, Abeyasekera S (2005) J Dairy Sci 88:2870–2878CrossRefGoogle Scholar
  39. 39.
    Poms RE, Glössl J, Foissy H (2001) Eur Food Res Technol 213:361–365Google Scholar
  40. 40.
    Poms RE, Hochsteiner W, Luger K, Glössl J, Foissy H (2003) J Food Protection 66:304–310Google Scholar
  41. 41.
    Rizzi A, Panebianco L, Giaccu D, Sorlini C, Daffonchio D (2003) Ital J Food Sci 15:499–510Google Scholar
  42. 42.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory Press, Cold Spring HarborGoogle Scholar
  43. 43.
    Schubbert R, Renz D, Schmitz B, Doerfler W (1997) Proc Natl Acad Sci USA 94:961–966CrossRefGoogle Scholar
  44. 44.
    Studer E, Dahinden I, Lüthy J, Hübner P (1997) Mitt Gabriele Lebensm Hyg 88:515–524Google Scholar
  45. 45.
    Thomas CM, Nielsen KM (2005) Nature Rev Microbiol 3:711–721CrossRefGoogle Scholar
  46. 46.
    Van den Eede G, Aarts H, Buhk H-J, Corthier G, Flint HJ, Hammes W, Jacobsen B, Midtvedt T, van der Vossen J, von Wright A, Wackernagel W, Wilcks A (2004) Food Chem Toxicol 42:1127–1156CrossRefGoogle Scholar
  47. 47.
    Vaneechoutte M, Young DM, Ornston LN, De Baere T, Nemec A, Van Der Reijden T, Carr E, Tjernberg I, Dijkshoorn L (2006) Appl Environ Microbiol 72:932–936CrossRefGoogle Scholar
  48. 48.
    Yonemochi C, Ikeda T, Harada C, Kusama T, Hanazumi M (2003) J Anim Sci 74:81–88CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Aurora Rizzi
    • 1
  • Lorenzo Brusetti
    • 1
  • Stefania Arioli
    • 1
  • Kaare M. Nielsen
    • 2
    • 3
  • Isabella Tamagnini
    • 1
  • Alberto Tamburini
    • 4
  • Claudia Sorlini
    • 1
  • Daniele Daffonchio
    • 5
  1. 1.Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM)Università degli Studi di MilanoMilanItaly
  2. 2.Genøk-Center for BiosafetyTromsøNorway
  3. 3.Department of PharmacyUniversity of TromsøTromsøNorway
  4. 4.Dipartimento di Scienze AnimaliUniversità degli Studi di MilanoMilanItaly
  5. 5.Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM)University of MilanMilanItaly

Personalised recommendations