Veterinary Research Communications, 27 Suppl. 1 (2003) 699–701© 2003 Kluwer Academic Publishers. Printed in the Netherlands

Detection of Genetically Modified Organisms (GMOs) in Food

and Feedstuff

E. Novelli1*, S. Balzan1, S. Segato2, L. De Rigo3 and M. Ferioli31Dipartimento di Sanita Pubblica, Patologia Comparata e Igiene Veterinaria,Universita degli Studi di Padova, 35020 L egnaro; 2Dipartimento di ScienzeZootecniche, Universita degli Studi di Padova, 35020 L egnaro; 3L aboratorio EPTANORD, 35026 Conselve, Italy*Correspondence: Dipartimento di Sanita Pubblica, Patologia Comparata e IgieneVeterinaria, Universita degli Studi di Padova, 35020 L egnaro, Italye-mail: enrico.novelli@unipd.it

Keywords: feedstuff, foods, genetically modified organisms, PCR

INTRODUCTION

Nucleic acid (DNA) amplification using polymerase chain reaction (PCR) allows theidentification of genetically modified organisms (GMO) in many kinds of food pro-ducts. One of the more widely used analytical protocols is reported in the Swiss FoodManual (1998), where DNA extraction is accomplished using WizardTM resin(Promega Corp., WI, USA), followed by a screening of DNA amplification in orderto search for the 35S promoter. Positive samples may then be submitted for specificPCR-assay for transgenic crop Bt-176 (Novartis) in the case of corn and Roundup-ReadTM (Monsanto) in the case of soybean crops. The analytical results reported hereare part of routine analytical tests required by the HACCP protocols or in purchasespecification control of raw materials and semi-finished goods supplied to food com-panies. It was, however, necessary to use two different extraction methods aimed atachieving the largest possible yield of DNA (while also ensuring the highest possiblepurity) in order to remove some causes of PCR inhibition.

MATERIALS ANDMETHODS

231 samples, provided by various agro industrial companies, were tested during 2001.Samples submitted for analysis, grouped into different categories, are shown inTable I. Two different techniques of DNA extraction were employed: the extractionmethod reported by Boom and colleagues (1990), modified by Tartaglia and col-leagues (1998), was employed in the case of foodstuff, feedstuff like ‘complete diet’,and a few samples of soybean. The method indicated in the Swiss Food Manual (1998)was used for the remaining samples (mainly maize and soybean for seeding). A PCR-assay screening was carried out in order to sift out GMO samples by means of

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TABLE IType of samples amalysed. Foodstuff means raw materials, semi-finished products and finishedgoods ( like baked foods and ‘polenta’). For feedstuff explanations see Table II

No. of % GMOSample samples % positive

Feedstuff 114 49.4 14.0Foodstuff 32 13.9 0.0Maize for seeding 85 36.8 0.0

promoter 35S identification (Pietsch et al., 1997). In samples of heterogeneous com-position the presence of amplifiable DNA was led using universal primers (Taberletet al., 1991). On the contrary, species-specific PCR assay was accomplished on maizeand soybean using target sequences zein (Studer et al., 1997) and lectin gene le1(Meyer et al., 1996). To avoid false positives, the fragment of 195 bp pertaining to the35S promoter, was submitted to restriction endonuclease digestion (XmnI).Quantitative analysis, using the Real-T ime technique adopting TaqManA chemistryperformed on thermocycler GeneAmpA 5700 (Applied Biosystems, Foster City, CA,USA), was performed on a few samples.

RESULTS

As reported in Table I, the largest group of samples tested was that of feedstuff andmaize for seeding. The category of foodstuff includes raw materials and semi-finishedproducts (baked products, different kinds of flour, ‘polenta’). All foodstuff samplesanalysed resulted negative for the 35S promoter, as were the samples under thecategory of maize for seeding. Table II shows results regarding feeds: two soybeansamples and eight ‘complete diet’ samples developed for cow nutrition, and all of thefeedstuffs for aviculture (six samples) were positive for the 35S promoter.

TABLE IIType of feedstuff samples submitted to analysis

No. of No. % GMOSample samples positive positive

‘Complete diet’ 88 8 9.1Feeds for poultry 6 6 100.0 (soybean RR)Soybean 2 2 100.0Other 18 0 0.0

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DISCUSSION

For some samples the routine utilization of the WizardTM method gave negativeoutputs for DNA amplifiability control. In such circumstances the extracted DNAshowed an anomalous coloration, subsequently giving a negative reaction to amplifi-cation by universal primers. On the contrary, the same samples submitted to DNAre-extraction by the use of the alternative method, gave positive results for the controlfor amplifiable DNA. Although both extraction techniques are based on the samechemical interaction (binding of nucleic acid on a bed of silica gel ), they differ fromeach other in the length time of sample incubation at 55–60°C, the type of reagentsused and the number of purification washings. In case of the WizardTM protocol,DNA adsorbed on the resin was washed once with 80% isopropyl alcohol as com-pared to the five washings used in the other method: twice with washing buffer(0.05 mol/L Tris-HCl, pH 6.4; 5 mol/L GuSCN), twice with 70% ethanol and finallywith acetone. The thoroughness in the washing procedure could be an explanationfor the removal of the cause of nucleic acid PCR inhibition. Therefore, consideringthe year in which they were developed it appears that speaking of HACCP programsand the checking of product specifications, the priority was the search for GMO byidentification of the 35S promoter fragment without any further discrimination forthe specific transgenic crop. In the case of feedstuff for poultry, the relative quota oftransgenic soybean was measured using specific primers. The quantity of transgeniccrop was variable from 0.37% to 11.32% in the six samples analysed. Soybean fromorganic agriculture and maize OGM-free certified (Other) were negative for the 35Spromoter or, at the least, contained levels under the sensitive threshold of the qualita-tive test.

REFERENCES

Boom, R., Sol, C.J.A., Salimans, M.M.M., Jansen, C.L., Wertheim-van-Dillen, P.M.E. and van der Noordaa,J., 1990. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology,28, 495–503

Meyer, R., Chardonnens, F., Hubner, P. and Luthy, J., 1996. Polymerase chain reaction (PCR) in thequality and safety assurance of food: detection of soya in processed meat products. Zeitschrift furL ebensmittel-Untersuchung und Forschung, 203, 339–344

Pietsch, K., Waiblinger, H.U., Brodmann, P. and Wurz, A., 1997. Screeningverfahren zur Identifizierung‘gentechnisch veranderter’ pflanzlicher Lebensmittel. Deutsche L abensmittel-Rundschau, 93, 35–38

Studer, E., Dahinden, I., Luthy, J. and Hubner, P., 1997. Detection of the genetically engineered ‘maximizer’maize using the polymerase chain reaction (PCR). Mitteilungen aus dem Gebiete derL ebensmitteluntersuchung und Hygiene, 88, 515–524

Swiss Food Manual, 1998. chapt. 52, Eidgenossische Drucksachen und Materialzentrale BernTaberlet, P., Gielly, L., Pautou, G. and Bouvet, J., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Molecular Biology, 17, 1105–1109

Tartaglia, M., Saulle, E., Pestalozza, S., Morelli, L., Antonucci, G. and Battaglia, P.A., 1998. Detection ofbovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials. Journal of Food Protection, 5, 513–518