Sensitive and highly specific quantitative real-time PCR and ELISA for recording a potential transfer of novel DNA and Cry1Ab protein from feed into bovine milk
- 417 Downloads
To address food safety concerns of the public regarding the potential transfer of recombinant DNA (cry1Ab) and protein (Cry1Ab) into the milk of cows fed genetically modified maize (MON810), a highly specific and sensitive quantitative real-time PCR (qPCR) and an ELISA were developed for monitoring suspicious presence of novel DNA and Cry1Ab protein in bovine milk. The developed assays were validated according to the assay validation criteria specified in the European Commission Decision 2002/657/EC. The detection limit and detection capability of the qPCR and ELISA were 100 copies of cry1Ab μL−1 milk and 0.4 ng mL−1 Cry1Ab, respectively. Recovery rates of 84.9% (DNA) and 97% (protein) and low (<15%) imprecision revealed the reliable and accurate estimations. A specific qPCR amplification and use of a specific antibody in ELISA ascertained the high specificity of the assays. Using these assays for 90 milk samples collected from cows fed either transgenic (n = 8) or non-transgenic (n = 7) rations for 6 months, neither cry1Ab nor Cry1Ab protein were detected in any analyzed sample at the assay detection limits.
KeywordsBovine milk ELISA MON810 Quantitative real-time PCR Validation
This study was funded by the Bavarian State Ministry of Nutrition, Agriculture and Forestry. The authors thank E. Englberger and T. Janke for their assistance. The staff at the Physiology Weihenstephan and the Bavarian State Research Center is gratefully acknowledged.
- 1.Hofte H, Whiteley HR (1989) Microbiol Rev 53:242–255Google Scholar
- 2.James C (2008) ISAAA Brief No. 37Google Scholar
- 3.(2003) Official Journal of the European UnionGoogle Scholar
- 4.Hupfer C, Hotzel H, Sachse K, Engel K-H (1997) Eur Food Res Technol 205:442–445Google Scholar
- 11.Poms RE, Hochsteiner W, Luger K, Glossl J, Foissy H (2003) J Food Prot 66:304–310Google Scholar
- 14.Swiss Food Manual (2004) Eidgenössische Drucksachen- und Materialienzentrale, BernGoogle Scholar
- 19.(2002) Official Journal of the European Communities L221Google Scholar
- 23.Hupfer C, Hotzel H, Sachse K, Engel K-H (1998) Eur Food Res Technol 206:203–207Google Scholar
- 26.Wiedemann S, Lutz B, Kurtz H, Schwarz FJ, Albrecht C (2006) J Anim Sci 84:135–144Google Scholar
- 31.Reuter T, Aulrich K (2003) Eur Food Res Technol 216:185–192Google Scholar
- 33.Shimada N, Miyamoto K, Kanda K, Murata H (2006) In Vitro Cell Dev Biol Anim 42:45–49Google Scholar