Abstract
The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA. A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)30. Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment. The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail. This allowed the subsequent determination of all hybrids through the use of the (dA)30-aequorin conjugate as a universal reagent. The bound aequorin was measured by adding Ca2+ and integrating the light emission for 3 s. As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2. The analytical range extended up to 2000 pM. As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2. The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5–19.8%. The use of a (dA)30-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.
Similar content being viewed by others
References
Erickson BE (2000) Anal Chem 72:454A-459A
Anklam E, Neumann DA (2002) J AOAC Int 85:754–756
Lipp M, Anklam E, Stave JW (2000) J AOAC Int 83:919–927
Stave JW (2002) J AOAC Int 85:780–786
Ahmed FE (2002) Trends Biotechnol 20:215–223
Holst-Jensen A, Ronning SB, Lovseth A, Berdal KG (2003) Anal Bioanal Chem 375:985–993
Anklam E, Gadani F, Heinze P, Pijnenburg H, Van Den Eede G (2002) Eur Food Res Technol 214:3–26
Bonfini L, Heinz P, Kay S, Van den Eede G (2002) Review of GMO detection and quantification techniques. European Commission Joint Research Center, Institute for Health and Consumer Protection, Food Products and Consumer Goods Unit, Ispra, Italy, pp 1–67
Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E (1999) J AOAC Int 82:923–928
Lipp M, Bluth A, Eyquem F, Kruse L, Schimmel H, Van den Eede G, Anklam E (2001) Eur Food Res Technol 212:497–504
MacCormick CA, Griffin HG, Underwood HM, Gasson MJ (1998) J Appl Microbiol 84:969–980
Meyer R (1999) Food Control 10:391–399
Garcia-Canas V, Gonzalez R, Cifuentes A (2002) J Agr Food Chem 50:1016–1021
Permingeat HR, Reggiardo MI, Vallejos RH (2002) J Agr Food Chem 50:4431–4436
Feriotto G, Borgatti M, Mischiati C, Bianchi N, Gambari R (2002) J Agr Food Chem 50:955–962
Mariotti E, Minunni M, Mascini M (2002) Anal Chim Acta 453:165–172
Jones K, Keenan M, Keenan M (1999) Trends Biotechnol 17:477–481
Shimomura O, Johnson FH (1978) P Natl Acad Sci USA 75:2611–2615
Head JF, Inouye S, Teranishi K, Shimomura O (2000) Nature 405:372–376
Kendall JM, Badminton MN (1998) Trends Biotechnol 16:216–224
Lewis LC, Daunert S (2000) Fresen J Anal Chem 366:760–768
Verhaegen M, Christopoulos TK (1998) Anal Chem 70:4120–4125
Laios E, Ioannou PC, Christopoulos TK (2001) Anal Chem 73:689–692
Glynou K, Ioannou PC, Christopoulos TK (2003) Protein Expres Purif 27:384–390
Glynou K, Ioannou PC, Christopoulos TK (2003) Bioconjugate Chem 14:1024–1029.
Trapmann S, Catalani P, Conneely P, Corbisier P, Gancberg D, Hannes E, Le Guern L, Kramer GN, Prokisch J, Robouch P, Schimmel H, Zeleny R, Pauwels J, van den Eede G, Weighardt F, Mazzara M, Anklam E (2002) Certified Reference Material IRMM-410S. European Commission Joint Research Center, Institute for Reference Materials and Measurements, Geel, Belgium, pp 1–20
Wurz A, Bluth A, Zeltz P, Pfeifer C, Willmund R (1999) Food Control 10:385–389
Acknowledgements
The financial support by the Secretariat of the Research Committee of the University of Athens and a Karatheodory research grant from the University of Patras are gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Glynou, K., Ioannou, P.C. & Christopoulos, T.K. Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe. Anal Bioanal Chem 378, 1748–1753 (2004). https://doi.org/10.1007/s00216-004-2507-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-004-2507-3