Qualitative detection of genetically modified material in crops and food products containing maize and soybean in Algeria

1 Laboratory of Phytopathology and Molecular Biology. École Nationale Supérieure d’Agronomie (ENSA), Algiers, Algeria. 2 Laboratory of Genetic Resources and Biotechnology, École Nationale Supérieure d’Agronomie (ENSA), Algiers, Algeria. 3 Centre National de Contrôle et de Certification des semences et plants. El Harrach, Algiers, Algeria. 4 Institut National de la Recherche Agronomique d'Algérie, El Harrach. Algiers, Algeria.


INTRODUCTION
In Algeria, the discussion around genetically modified organisms (GMO) regulation and other related issues appeared with the adoption of the Cartagena Protocol on biosafety, although the country had only little experience in modern biotechnology and biosafety regulation (Louanchi, 2009).Agricultural development in the country, a fossil fuel producer, has not been prioritized for several years.For that reason, most basic food and feed, such as cereals (wheat, maize) and oil-yielding plants (canola, soy) are imported as unprocessed or transformed products.The concern of becoming a passive consumer is even greater in light of recent studies on animal health (Séralini et al., 2014).
Moreover, introduction of the first hybrids in the 1990s failed increasing yields and provoked a genetic erosion of landraces and during the last decade, the agricultural sector showed an important development mainly based on massive importation of agricultural inputs like seeds  , 2000).However, there is no specific regulation to control the presence or use of GMOs in food crops and feed, as well as no regulation for food labelling.This situation prevents us from getting insights on the presence or absence of GMOs in agriculture, food and feed chains.Earlier studies on GMOs identification have been carried out by using serological and polymerase chain reaction (PCR) detection methods and the results confirmed the presence of GMOs in several analyzed samples (Ghazi, 2007;Louanchi et al., 2008).Screening methods for qualitative detection (that is, presence or absence of a transgenic event) allow rapid detection of a great number of GMOs.It is a simple and fast method based on differential amplification of specific sequences commonly found in many transgenic events (Wolf et al., 2000), but does not allow differentiation of the involved GMO.In general, the screening of the 35S promoter from Cauliflower Mosaic Virus (P35S CaMV) and terminator of the Agrobacterium tumefaciens nopaline synthase gene (T-Nos) allows the detection of most of the authorized GMOs (Hemmer, 1997).
The aim of this study is (i) to ensure that soybean and maize seeds introduced in Algeria are not genetically modified and (ii) to test local and imported maize and soybean for the presence of GMOs in the food and feed chain in Algeria.

DNA extraction and purification
All the procedures were carried out under sterile conditions.Soybean and maize samples were ground with an electric grinder.40 mg of the resulting flour were used for the extraction of the genomic DNA according to the method described in the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA).The extracted DNA pellet was air dried and dissolved in 50 μl of sterile distilled water.The concentration and purity of the extracted DNA were measured by absorbance under UV at 260 and 280 nm, using a spectrophotometer (NanoDrop 2000 ® Thermoscientific).The DNA concentration was adjusted to 25 ng/μl prior to PCR by diluting into sterile distilled water.

Oligonucleotides primers used
Primers were used to amplify DNA sequences of specific genes from maize (Studer et al., 1998), soybean (Meyer et al., 1996), the promoter 35S of Cauliflower Mosaic Virus (CaMV) (Wurz and Willmund, 1997), and were used to detect Roundup Ready GM crops (Elsanhoty, 2013) and also several Bt Maize, and the Terminator Nopaline Synthase (T-Nos) (Lipp et al., 2001) mostly observed on transgenic events (Table 2).All primers were synthesized by DIAG-GENE (Angers Technopole, France).Before their use, lyophilized primers were diluted in purified water to obtain a final concentration of 20 pmol/μl.

DNA amplification and PCR conditions
PCR amplification was performed on a C1000 Thermal Cycler (BIORAD).Amplification conditions for each set of target sequences, as described by Querci et al. (2006), are presented in Table 3.Each PCR reaction mix (25 μl total volume) contained 2.5 μl PCR buffer (10x concentrate, Promega), 2 μl MgCl2 solution (25 mM MgCl2), 1 μl dNTP solution 0.2 mM of each of dATP, dCTP, dGTP and dTTP, 0.5 μl of each primer, 1 Unit AmpliTaq Gold DNA amplicons were separated on 2% w/v agarose gels (Promega) using TBE buffer solution at pH 8.0 (0.89 M Tris, pH 8.0; 0.89 M Boric acid; 2 mM Na 2 EDTA) and 0.5 µg.ml -1 of ethidium bromide at 150 v for 45 min.In addition, amplified target fragments of transgenic P35S CaMV and T-Nos sequences (DIAG-GENE, Angers Technopole, France) used as positive controls, were loaded on the gel.Target DNA sequences were observed with UV transilluminator Gel Doc Molecular Imager (BIORAD).

Amplification of extracted DNA
In order to assess the presence of transgenic elements, DNA extraction using Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) allows us to obtain a DNA of a good quality to perform PCR amplifications.PCR quality control, as recommended by the EU procedures for GMO detection (Querci et al., 2006), was performed with the specific primers for maize and soybean DNA.The primer pair GM 03/GM 04 is specific for the single copy lectin gene LE1 and yields a PCR product of 118 bp size (Meyer et al., 1996) detectable in transgenic as well as in conventional soybean.The primer pair ZEIN3/ZEIN4 is specific for the zein gene and yields a PCR product of 277 bp size (Studer et al., 1998) detectable in transgenic, as well as in conventional maize.Soybean and maize specific primer pairs served as a control for the amplification of the isolated DNA and PCR procedure (PCR quality control).All the tested samples gave positive results for the amplification of the control primer pairs.These results confirmed that the samples from processed food, like flour or meal, are based on maize or soybean.

DNA target sequences detection
Primers p35s-f2 and petu-r1, specific for the detection of the 35S promoter of CaMV, amplified a 172 bp fragment.These fragments are only detected in GMO samples.A specific band was amplified for the 35S promoter in 29 out of the 120 samples tested (20 from maize and 9 from soybean) suggesting that these samples contain GMO (Figure 1).Similarly, primers HA-nos118-f and HA-nos 118-r, specific for the detection of terminator nos, amplify a 118 bp fragment, detected only in transgenic samples.Here also, a specific band was amplified for the nos terminator in the same 29 samples, therefore corroborating the presence of a transgenic event (Figure

Investigated transgenic samples
During this experiment, 120 samples of maize and soy were analysed for the presence/absence of GMOs.For crops, samples were represented by ears (43) of maize originating from several fields in eleven different localities, maize seeds (7 varieties), and one seed variety of soybean.For food products collected from markets and harbours, samples were represented by maize as food grain (18), soy as food grain (2), canned maize (21), corn flour (5), popcorn ( 12) and soybean flour (11).Twenty nine samples (20 from Maize and 9 from Soybean) showed the specific DNA fragment either for P35S CaMV fragment or T-Nos fragment.
Results show that transgenic elements are not present in the agricultural products (ears and seeds) investigated.This reveals that the Ministerial Decree of 2000 prohibiting the imports of GMO containing material for agricultural uses is effective.Although these crops are not widespread in Algeria, their use as animal feed and processed food goods is wide spread, therefore, we believe that it is important to strengthen this decree by setting up laboratories of control and validation of standard methods for detection as it is done in several countries, especially for UE legislation (Trapmann et al., 2001(Trapmann et al., , 2002)).
Indeed, it was found that four samples from grain food (3 of maize and 1 of soybean), which are close to seeds in terms of propagation, were detected positive for GMOs in our studies.Although, they are designated for food transformation, these grain feed had more than 90% of germination rate (laboratory data).This result highlights a crucial issue concerning the denomination of GMO or LMO (living modified organisms) and suggests that the latter should be included in the risk assessment, because of their possible dissemination.
Over 50% of the GMO positive samples (16 out of 29) were imported from Latin America (mainly from Argentina) as food grain, popcorn or canned food (Table 4).Six samples were imported from Asia (Thailand and China) as canned maize.Two were from USA as food grain, while five were of unknown origin.

DISCUSSION
The present studies reveal that GMOs are not present in seeds in Algeria.This result is important regarding the Algerian regulation restricting the use of GM material.However, the regulatory system should evolve towards a strengthening of the control mechanisms, including a Food grain Unknown better regulation on GM related administrative procedures, risk assessment guidelines, implementation of efficient laboratories for GM control using more sensitive methods such as semi-quantitative detection (Tozzini et al., 2000), and real-time quantitative PCR detection (Poitras and Houde, 2002;Yang et al., 2007) with validated and standardized methodologies.
Our results showed that several food products which contained GMOs are mostly imported from Argentina, Thailand and China and this situation raises the problem of labelling and traceability (Miraglia et al., 2004).Indeed, the lack of labelling regulation is a concern for numerous countries, which could be restrictive countries like Saudi Arabia (Abdel-Mawgood et al., 2010) or Tunisia (Chaouachi et al., 2013) or permissive countries regarding GMOs content like South Africa (Viljoen et al., 2006) and Egypt (Tony et al., 2003).
This work is the first contribution in Algeria on the presence of GMO in crops and food products.Results show the absence of GMO in crops and seeds but their presence in 29 samples of foodstuffs.Further studies are needed to quantify the amount of GMOs that are present in the food products throughout the Algerian market.For that purpose, genetically modified products in food commodities should be checked and controlled to comply with the EU and international regulations that enforce labelling of food when GMO presence is higher than 0.9%.Although some countries might be tolerant with GMOs presence in food, the precaution principle is thought to be applied for as long the risk for human health on the long run is not known.
Our results suggest that, to comply as well with the national regulation, genetically modified material detection and monitoring plans are necessary to control the distribution of unlabelled GM-containing foods and feed in the Algerian market.
For an effective management of the genetically modified organisms and in order to set up a national policy which can face the transnational movement of both agricultural and food product, Algeria must set up a regulation which include labelling and traceability and a detection methodology with homogenized protocols and adapted techniques used in routine.

Table 1 .
Presentation of maize and soy products analysed for GMO PCR detection.

Table 2 .
DNA primers used and corresponding target DNA sequence.

Table 3 .
PCR conditions for all target DNA sequences.
polymerase (Promega), 5 μl of template DNA and completed to 25 μl with purified water.Negative control presented the same PCR reaction mix with water instead of extracted DNA.All amplified products were stored at 4°C until the gel electrophoresis.

Table 4 .
Presentation of GMO samples detected and their origin.