Presence of Bacillus thuringiensis ( Bt ) gene in cereals and cereal-based products in local markets and supermarkets of Yaoundé , Cameroon

The use and importation of genetically modified (GM) crops and products derived from these crops are regulated by national and international policies, which unfortunately are often not properly implemented in some countries. Given the ongoing globalization of trade and increasing availability of GM plant products, countries like Cameroon with a weak system to regulate the importation of these products face the threat of these products entering local markets. This study investigated the presence of GM cereals and cereal-based products circulating in the local markets and supermarkets in Yaoundé, Cameroon. An inventory of cereal based products from these markets was conducted and one of the products was labeled as being derived from GM cereals crops. DNA was extracted from 26 products with a protocol using SDS guanidine thiocyanate to assess the presence of the Bacillus thuringiensis (Bt) gene. Polymerase chain reaction (PCR) was used to amplify the DNA fragment associated with Bt gene. Majority of the products were maize-soya based and wheat-soya based. The Bt gene was present in four of the 14 maize based products tested. The presence of the Bt gene in these cereal based products suggest the need for these products to be labeled according to international regulations.


INTRODUCTION
The exponential growth in human population during the last century has left various challenges, especially in the domain of feeding and health in many countries.Given this rapid growth and some environmental and agricultural challenges in many countries (Kishore et al., 1999), much effort was needed to address threats that impeded crop production.Traditional and modern biotechnology has been used to improve agronomic traits in plants.As reported, one advantage of growing genetically modified (GM) crop is the reduction in the use *Corresponding author.E-mail: wfmbacham@yahoo.com.Tel: +237677579180.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Ivr 1A = Primer for the forward strand and Ivr 1B = primer for the reverse strand of the invertase gene; Cry 1As = primer for the forward strand and Cry 1Ab = primer for the reverse strand of the Bt gene.
of chemicals to control pests, leading to increased productivity and profitability (Halford et al., 2000;Engel et al., 2002;Gomez-Barbero et al., 2006).However, many concerns have been raised on the use of GM crops.The negative consequences of the widespread use of GM plants have been divided into two categories involving: (1) adverse effects on the environment and (2) harmful effect on human and animal health (Halford et al., 2000;FAO, 2003;Qaim, 2009).The most commonly cited risks have been transfer to nontarget species (Halford et al., 2000;Cartagena Protocol on Biosafety, 2000) and the possible long term undesirable consequence of biodiversity erosion through the loss of traditional crops (Firbank et al., 2006).
The negative socioeconomic effects caused by the corporate control of the food chain have also been reported.Considering the dominance of the biotech industry by large multinational corporations (MNCs), the shift in seed control from a local to a corporate level reduces farmer's involvement in seed enhancement as well as control over "food sovereignty" (FA0, 2003;Azadi et al., 2015).Considering the controversy, policies have been proposed to insure a free and informed adoption and control of genetically modified products (Hogan et al., 2001;Nair et al., 2002;MINEP, 2003;Vacher et al., 2009).Before the introduction of these products in Africa, ethical, cultural environmental, health, economic and ecological concerns were raised (MINEF/UNEP/GEF, 2003).Today, African countries have adopted GM products for commercialization (James, 2008).
Cameroon has ratified the Cartagena Protocol on Biosafety, but has not yet adopted GM crops for commercialization.Given the globalization of trade, the wide-spread adoption of GM crops, and the weak system to regulate GM food products in Cameroon, it was necessary to identify such foods in the market.This study investigated the presence of GM cereals and cerealbased products circulating in the local markets and supermarkets in Yaoundé, Cameroon.

Sample collection and preparation
An inventory of maize, wheat and soya based products or combination from local markets and supermarkets was carried out using a structured questionnaire.Data recorded included the manufacturer, expiry date, manufacturing date, ingredients of the product, composition of the product, physical form of the product, place of manufacturing and packaging form.Processed products and seeds were collected for maize and soya-based products.The maize grain collected from the field was used to standardize the protocol for DNA extraction.Non-powdered products were separately milled and 30 g of each sample was placed in a plastic bag, sealed and stored at 25°C in a dry place.

DNA extraction
Total genomic DNA was extracted as indicated by Edwards et al. (1991) for the cell lysis steps and Chomczynski and Mackey (1995) for protein and nucleic acids precipitation steps.Note that, the cell lysis solution was added to the mixture followed by proteinase K (20 mg/ml).After an overnight incubation of this mixture, RNAse H 250 UI was added followed by incubation at 37°C, as the objective was the extraction of total DNA.The obtained DNA pellet washed using 70% ethanol with the pellet air-dried, was re-suspended in 200 µl of 1xTris-EDTA buffer.DNA samples were stored at -20°C until further analysis.

The polymerase chain reaction and optimization of reaction conditions
Amplification profiles for the invertase and Bt genes were established through two different PCR procedures; the touchdown PCR and the multiplex PCR.These methods were applied to determine the best annealing temperatures.The multiplex PCR was used to check if the two genes could be amplified under the same conditions.The method of Brinegar et al. (2004) was used with modifications.The PCR mixture consisted of 4 µl of DNA extract, 0.25 µl of 5 U/ml Taq polymerase, 0.25 µl of each primer (Table 1), 0.5 µl of 10 mM dNTPs, 2.5 µl of 10x thermopol buffer in a final volume of 16 µl.The amplifications were performed using a T3 thermal cycler (Biometra, UK) with the following conditions: 3 min at 94°C (pre-denaturation), 45 cycles of 45 s at 94°C (denaturation), 45 s at 69°C for Ivr gene and 50°C for Bt gene (annealing), and 30 s at 72°C (elongation), with a terminal step of 5 min at 72°C.The mixture was held at 4°C.

Electrophoresis of PCR products
PCR amplification products were separated by gel electrophoresis using 2.5% agarose gels (Seakem) with 1×Tris-Borate-EDTA (TBE) running buffer and stained with ethidium bromide.DNA fragments were visualized on an ultraviolet trans-illuminator and photographed with a digital camera.

Source and characteristics of study samples
Samples collected were from various places of manufacture (Table 2).Four products were collected from local markets and 22 from supermarkets (Tables 3 and 4).

DNA extraction and PCR assay
DNA was successfully extracted from 14 products using SDS and guanidine thiocyanate.Amplification of the Ivr gene in a sample was used as an indicator for a successful DNA extraction procedure.DNA was not successfully extracted from wheat-based products.
Invertase gene was used as an internal positive control for DNA extraction and PCR conditions.Only Ivr-positive samples were tested for Bt-gene.Four of the 14 products tested were Bt-positive (Figure 1).The product used to standardize the protocol for DNA extraction was also tested for Bt gene and was found positive.Band sizes for amplified Ivr gene and Bt genes were 240 bp for Ivr gene and 200 bp respectively (Figure 2).Ivr-positive products were maize and soya based products.No amplification of Ivr gene was obtained with wheat-based products and multi-cereal products.Bt gene was mostly found in maize-based products.

DISCUSSION
Cameroon, although it has ratified the Cartagena protocol on biosafety, has not yet adopted GM crops and derived foods for commercialization.Actually, the quality control scheme of GMOs and derived products is being built.In this process, a comprehensive organization is needed.
Laws have been enacted but yet few are implemented.
For instance, it is accepted globally and recommended that GMOs and derived products should be labeled to insure a free and informed consumer choice.The presence of just one sample label that conforms to this law reveals the limited practical implementation of the regulation in use in Cameroon (Decree No. 2007/0737/PM of the 31 st May 2007).Even though each country or region need to establish threshold value for GM-based product labeling application, Cameroon and even the Central region of Africa has not yet defined the percentages at which a product can be label as containing genetically modified ingredients.It might mean that no matter the percentages of ingredients present in a food product, according to the law, it should be labeled.
In the process of food control, laboratories analyses play major role as quality results are needed for good and right decisions to be taken in the regulatory processes (see "Elements for a National Biotechnology Policy Framework for Cameroon").Using PCR techniques, the purification of nucleic acids from the sample is often the deciding factor in the production of meaningful results.
The combined protocol used in this study can be used with success for DNA extraction from various food types.However, the method was best suited for isolation of DNA from less processed foodstuffs such as corn flour and corn seeds.Improvements are needed to isolate DNA from highly processed foodstuffs that might also contain PCR-inhibitory substances.This was the case of wheat-based products which consistently failed (Tengel et al., 2001).It will also be of use to evaluate the power of the method in terms of quantities and quality of DNA obtained.This will allow the establishment of standards for each product type.Successful amplification of Bt-gene in cereals and cereal-based products made in Cameroon suggest the use of Bt maize seeds by farmers and in the industry; the source and origin are still to be identified.However, the identification of Bt-positive products was based on the qualitative-PCR analysis using species-specific primers.The obtained molecular sizes were not very different from those reported in the literature (Tengel et al., 2001;Brinegar et al., 2004).Coupled with other techniques as the quantitative-PCR analysis, Western blot, the method used in this study would allow a broad application (Roger et al., 2014).Amplification of the Ivr and Bt genes fragments demonstrated that DNA of integrity sufficient for PCR analysis can be purified using the described techniques.
The finding of this study shows the need to strengthen the national bodies in charge of control at different levels, with capacities and equipments for an efficient and effective action.Given the role of laboratory analysis in the established regulation processes for GMOs, laboratories should be equipped with appropriate trainings and equipments.A broad range assessment of various food types in their various forms would allow a global view at national scale and the standardization of laboratory techniques for GMO detection in Cameroon.As Cameroon is considered a leading country in the Central region of Africa, the presence of National Laboratories for GMOs testing would be a valuable asset in the northern region and in the southern part of the country, as these laboratories would serve for the Central Africa zone.Quality control for GMOs testing should be added to the quality control system of the country at the various customs posts, especially at the national ports.

Conclusion
The finding of this study indicates that many of the maize-, wheat-and soya-based products present in local markets and supermarkets in Yaoundé are not labeled according to the regulation in use for genetically modified products in Cameroon.Many of which are GM-derived

A B
food products; it will be necessary for the Cameroon government to revise this regulation to meet this challenge.

Figure 1 .
Figure 1.Bands for Ivr gene using the two different annealing temperatures (PNa=74°C -PNb=69°C).-, No bands for Bt gene with the annealing temperature of 69°C; MM = 100 base pair molecular weight marker (MM); PNa1 = PN sample code; a = annealing temperature of 69; 1 = the first tube.

Figure 2 :
Figure 2: PCR fragments for Ivr (A) and Bt (B) genes

Table 1 .
Sequences of primers used in this study.

Table 2 .
Sample distribution per manufacturer/country.

Table 3 .
Sample distribution per type of cereals.

Table 4 .
Distribution of Ivr and Bt-genes among the study samples.

Table 2 :
Sample characteristics per manufacturer/country (list of sample attached to this document)

Table 3 :
Sample distribution per type of cereals present