application of genetically modified crops: status...

Application of Genetically Modified Crops: Status, Regulation and Detection Method in Indonesia

Bahagiawati dan Sutrisno

Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Jl. Tentara Pelajar 3A Bogor-16111

ABSTRACT

Application of Genetically Modified Crops: Status, Regulation, and Detection Method in Indonesia. Bahagiawati and Sutrisno. Global area of transgenic crop was increase tremendously. The number of country accepting of planting and/or marketing the transgenic crops and its derivative products also become more numberous. However, due to existing controversy on the benefit and risk, the application of transgenic crops was governed by regulations to protect the consumer and environment from its unwanted effects. There are some international conventions that managing and controlling the uses of these crops, one of them was Cartagena protocol that Indonesia ratified in 2004. Indonesia also launched a regulation upon labelling package food derived from transgenic crops in 1999. To implement either the Cartagena protocol and labelling regulation, Indonesia needs to increase its capacity to detect the present of the transgenic crop product either in raw, and proceesed food. This review will discuss about the development of the application of transgenic crop and its product globally, and list of transgenic crops that have been accepted and approved as safe for human consumption and environment. The regulations upon the application of transgenic crop in Indonesia also be informed. Some metodologies to detect the presence of the genetically modified food that are generally use in some countries also be discussed in this review.

Key words: Transgenic crop, regulation, detection method, laboratory.

INTRODUCTION

One of the alternative technologies that can be used in plant breeding program to increase agricultural production is genetic engineering. Due to this technology, there was an increase in transgenic plant cultivation for commercialization. When introduced in 1996, transgenic plants were cultivated in 1.7 ha area and increased by 40 times to 67.7 million in 2003 (James 2003). A third of that total area (20 million ha) is in developing countries. In 2003, more than 10 million farmers from 25 countries had planted transge-nic plants with the total market value of US$ 4 trillion. Those countries include USA (42.8 million ha or 63%), Argentine (13.9 million ha or 21%), Canada (4.4 million ha or 6%), and South Africa (0.4 million ha or 1%)

Hak Cipta © 2007, BB-Biogen

(James 2003). In 2006, about 19 kinds of transgenic plants were approved either for field released, human consumprtion and for feed. They are soybean, maize, canola, carnation flower, lentil, sugar beet, sunflower, wheat, linseed, melon, chicory, bentgrass, potato, papaya, rice, squash, tobacco, tomato and alfalfa (Agbios 2006). The increase of transgenic plant cultivation is followed by increase in their distribution, whether as raw materials such as seeds, as half-processed food or as food product in global market.

The presence of transgenic plants and their products has triggered public controversial reactions. One of the reasons is that genetically engineered products have not been tested, as opposed to conventional products that have been used for years. However, since the marketing of transgenic plants in 1996, the product had been accepted because, as stated by several countries, several transgenic products in markets are safe for human consumption and feed. Transgenic plant products have been approved in several countries, the approval stating that the products are save for environment, human consumption and feed. Several plant commodities and countries that released approval of transgenic crops are shown in Table 1.

Concern about negative effects resulting from transgenic products has caused several countries to produce, approve and implement regulations that guarantee the safe-utilization of these modern technology products so that they will not endanger consumers and will not damage natural resources. To protect consumer’s rights to choose the origin and type of products to be consumed, regulation about product labeling was made. Several countries that have this labelling regulations are shown in Table 2.

To implement the labeling regulation of genetically modified organism (GMO) products, the government needs to have capacity to detect whether a food product is originated and contained GMO. So, the methods for GMO detection need to be developed. The methods not only quickly, but also regional or international accepted. There are more than 100 mothods developed globally, and standardization of the methods have not been made until now. However, there are some effords to standardize these methods

Jurnal AgroBiogen 3(1):40-48

2007 BAHAGIAWATI AND SUTRISNO: Application of Genetically Modified

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through Codex and ISO. Since the end of 1990s several methods has been published by laboratories across countries. Because there is not yet international standard, several countries developed their own methods and used them as national guidelines.

The objectives of this review are to elucidate the transgenic plant status, products and regulation in Indonesia and the methods that are commonly used in the worlds to detect GMO.

TRANSGENIC PLANT STATUS, PRODUCTS AND REGULATION IN INDONESIA

In year the of 2001, for the first time Indonesian government approved environmental released of transgenic plant for commercialization, which was Bt cotton (Bollgard) in 7 districts in South Sulawesi. It did not take a long time for the National Biosafety Commission to give the approval for this comodity because the product was not consumed as food. The company who own the Bollgard sold their Bt-cotton seeds for only two years to the cotton farmers in South Sulawesi. However, due to several reasons, in the third year (2003) the company decided not to renewed the

Table 1. List of approved GMO for safety to use for human consumption and the countries that gave the approval.

Commodity Introduced trait No. of Event*

Countries that approved safe for human consumption

Alfalfa Herbicide tolerant 1 Canada, Mexico, Phillipine Argentine canola Herbicide tolerant 15 USA, Canada, Japan, China, European Union, Australia Poland canola Herbicide tolerant 2 Canada Cotton Pest resistant, herbicide tolerant 15 USA, Canada, Australia, Argentine, China, India, Japan,

Mexico, Phillipine, South Africa, Brazil Linseed Herbicide tolerant 1 USA, Canada Lentil Herbicide tolerant 1 Canada Maize Herbicide tolerant, pest resistant, fertility recover,

male sterility, lysine-content intensifier 32 USA, Japan, Canada, Phillipine, Taiwan, China, Argentine,

Australia, European Union, South Africa, Netherland, UK, Switzerland, South Korea, Rusia, Uruguay

Melon Delay ripening 1 USA Potato Pest resistant, herbicide tolerant 4 Australia, Canada, Japan, Phillipine, USA Rice Herbicide tolerant 3 Canada, USA Soybean Herbicide tolerant, oil content 7 Australia, USA, Argentine, Brazil, Rusia, UK, Taiwan, Japan,

South Africa, Uruguay, Mexico, European Union, Switzerland, South Korea, China

Squash pumpkin Virus resistant 2 Canada, USA Papaya Virus resistant 1 Canada, USA Sugar beet Herbicide tolerant 3 Canada, USA, Phillipine, Australia, Japan Sunflower Herbicide tolerant 1 Canada Tomato Delay ripening, pest resistant 6 Canada, USA, Japan, Mexico Wheat Herbicide tolerant 5 Canada, USA

* Approved safe for human consumption. Source: Agbios (2006).

Table 2. List of countries possessing labeling regulation.

Country Labeling procedure Threshold Label

Australia and New Zealand Mandatory 1,0 Genetically modified Brazil Mandatotry 1,0 Genetically modified Canada Voluntary 5,0 Non-Genetically modified China Mandatory 1,0 Genetically modified European Union Mandatory 0,9 Genetically modified Indonesia Mandatory 5,0 Genetically modified Israel Mandatory 0,9 Genetically modified Japan Mandatory 5,0 Genetically modified Phillipine Voluntary n/a Genetically modified Rusia Mandatory 0,9 Genetically modified Saudi Arabia Mandatory 1,0 Genetically modified South Korea Mandatory 3,0 Genetically modified Switzerland Mandatory 0,9 Genetically modified Taiwan Mandatory 5,0 Genetically modified Thailand Mandatory 5,0 Genetically modified USA Voluntary n/a Non Genetically modified and organic

Source: Viljoen (2005).

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annual released approval of their Bt-cotton in Indonesia (Bermawie et al. 2003).

By unintentionally, some transgenic plant products were distributed in Indonesia for several years. To fulfill the need for food, Indonesia imports soybean and maize from abroad. For example, in 2003, Indonesia imports about 2.5 million ton soybean because the national soybean production was only 672.4 thousand ton (BPS 2004). The imported soybean was largely (70-90%) obtained from the USA and Argentine which are the main transgenic cultivating countries in the world (Swaco Prima Windutama, 2005). Similar import happened with maize. Even though Indonesia produces maize, the national production is not enough to supply the demand of Indonesian population. These days, 43% of the maize demand is for human food supply, and the rest is for feed (Kasryono 2002). Indonesia imported 700 thousand ton maize mainly from the USA and Argentine in 2004 (Swaco Prima Windutama 2005). Therefore, the presence of transgenic plant products in Indonesia is unavoidable since last couples of years.

Indonesia has some regulations that control the utilization of transgenic plants such as Regulation no. 7 year 1996 about food and Regulation no. 21 year 2005 that revised prior laws. In addition, since 1999 Indonesia has released regulations about GMO product labeling with Regulation (president Order) no. 69 year 1999. In 2004, regulation about genetically engineered food product was released in Regulation no. 28 year 2004. However, Regulation no. 69 year 1999 and no. 28 year 2004 have not yet been implemented, because the guideline was not launched yet.

Until now, several applications for commercial released of transgenic plant have been submitted to

the National Biosafety Commission National Biosafety Commission (Table 3). Some of the applications were under reviewed.

In 2004, in an attempt to harmonize the methods and sharing experiences and capability an ASEAN (Association of South East Asian Nation) GM Food Testing Network was formed with the name of ASEAN Genetically modified Food Testing Network, which as initiated and coordinated by Singapore, and Indonesia is a member. Meeting is held annually, while the members take turn to be the host.

To date, Indonesia only has four laboratories that run GM Food detection. Two belongs to the government, ICABIORAD (Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development) under the Department of Agriculture and Badan POM (National Food and Drugs Control). The other two are private owned, PT Saraswanti and University of Atmajaya (Table 4).

METHODS FOR DETECTION OF FOOD MATERIALS AND PROCEESSED FOOD CONTAINING GMO

Before going into detail on the detection methods, it is necessary to know the genes and traits that are commonly found in transgenic plant those are found in markets globally. These data are important to determine the target gene that will be detected. In addition, this paper also will explain the need for using reference material in the methods.

Genetically Engineered Transgenic Plants

Transgenic plants are plants resulted from introduction of DNA fragment of other organism into the plant genome. The process is knows as

Table 3. Status of GMO analysis on biosafety and food product at Biosafety Technical Team level in 2006.

Plant Introduced trait (plant name)

Proponent (year dossier submitted)

Biosafety containment Field confine Multilocation Results Status

Insect resistant (MON810)

Dupont (2002) done Waiting for approval

Herbicide tolerant (GA-21)

Monsanto (2002) done done environment safe Waiting for approval

Maize

Herbicide tolerant (NK603)

Monsanto (2002) done done Waiting for approval

Cotton Cotton fruit borer resistant (Bollgard)

Monsanto (1998) done done done Released in 2001, stopped in 2003

Soybean Herbicide tolerant (GTS 40-3-2)

Monsanto (2002) done done Waiting for approval

Rice Stemborer resistant (Bt-rice)

LIPI (2003) done Undergoing In process of data collection for biosafety

Sugarcane Drought tolerant (sugarcane bet A)

PTPN XI (2003) done Undergoing In process of data collection for biosafety

None of the above commodities have been assesed for food safety. Source: Bahagiawati et al. (2006).


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transformation. DNA fragment that is integrated into the plant genome is usually obtained from organisms present in nature such as bacteria or other plants. The gene construct that is introduced into the host plant usually contains three components, which are (1) promoter that functions to activate or deactivate the introduced gene, (2) the introduced gene that will express desired trait, and (3) terminator that stops reading signal from the gene sequence (Viljoen 2005). Several promoters that are commonly used to produce transgenic plant, but the one that are frequently used is P-35S from Cauliflower Mosaic Virus. The terminator that commonly used is T-NOS from Agrobacterium tumefacien.

Nearly all commercialized transgenic plants contain P-35S promoter and T-NOS terminator. Therefore, P-35S and T-NOS sequences are often used to screen GMO. However, screening in this way often results in false positive that comes from virus or bacterium that are present in the plant sample.

Even though some GMO contain identical insert, promoter and terminator, they are considered different transgenic plants (with different events) and are named differently because they are unequal in the plant genome location where the genes are inserted.

Commonly Transgene in GMO

Transgenic plant resistance to insects is due to introduction of cry genes (cry1Ab, cry1Ac, cry1F, cry2b, cry3A, cry3Bd1, and cry9c) from several soil bacterial subspecies B. thuringiensis (Viljoen 2005). cry genes encode “Bt” toxin, a delta endotoxin that can be toxic to several species of insects, including plant pests. This toxin binds to the cell receptor in the digestive track of the insect, disturbing the ion flux, which causes dysfunctional digestive track and death of the insect. Mammals do not have the same type of receptor in their digestive track as the insects; hence the presence

of Bt toxin will not disturb the human digestive system (Hofte and Whiteley 1989).

There are two types of herbicidal tolerance in transgenic plants according to the active ingredients of the herbicides. They are glyphosate (commonly known as Roundup Ready) or phosphinothricin (Viljoen 2005). Glyphosate inhibits essential amino acid synthesis in plants, especially aromatic amino acids phenylalanine, tyrosine, and tryptophane by as amino acid analogue that inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Herbicide-tolerant transgenic plants contain modified form of EPSPS obtained from Agrobacterium tumefaciens to make the plant tolerant to glyphosate. This gene is also used together with the gene encoding glyphoste oxidoreductase (GOX) that inactivates the glyphosate. The other herbicide-tolerant transgenic plants relate with phosphinotricin. The active ingredient of phosphinothricin is glufosinate ammonium that can inhibit glutamine synthase and cause the accumulation of ammonia and kill the plant. The gene is isolated from soil bacterium Streptomyces viridochromogenes that encodes phosphinothricin-N-acetyltransferase (PAT) that inactivates glufosinate. This gene is used to weed control.

Several PCR methods for some transgenic maize that are commonly found have been published, such as transgenic plant detection method for maize like transgenic Bt11, MON810, event186, T25, and GA-21. The target genes from transgenic maize above are shown in Table 5.

Certified Reference Materials

Reference materials that offer reliable positive and negative controls provide basic validation to procedural analyses and performance of methods and laboratories. Every type of GMO needs specific reference materials. DNA extracted from GMO plants,

Table 4. Indonesian laboratories that perform GMO detection test.

Institution Type of testing Proficiency test followed

ICABIOGRAD Laboratory Qualitative Indonesian Accreditation Committee (KAN)-2003 Asia Pacific Laboratory Accreditation Cooperation/APLAC-China (2004)

National Food and Drug Control Laboratory Qualitative Asia Pacific Laboratory Accreditation Cooperation/APLAC-China (2004) ASEAN-Malaysia (2005)

Saraswanti Laboratory (private) Qualitatif and partly quantitative Indonesian Accreditation Committee (KAN)-2003 Central Science Laboratory (CSL)-UK (2003) Asia Pacific Laboratory Accreditation Cooperation/APLAC-China (2004)

Universitas of Atmajaya Biotechnology Laboratory (private university)

Kualitatif Not yet accredited and never taken proficiency test

Source: Bahagiawati (2006).


seeds, plasmid/vector constructs and pure protein can be used as reference materials. The use of DNA from seeds as reference materials portrays natural situations because it removes matrix effects, while plasmid and protein can be prepared in large amount and more consistent.

The availability of reference materials is very limited due to Intellectual Property Right (IPR). The presence of Institute of Reference Materials and Measurements (IRMM) of Joint Research Center (JRC) in Geel, Belgia, provides several certified reference materials that can be bought through FLUKA (Buchs, Switzerland) and AOCS (Illinois, USA) for transgenic soybean, maize and others (Pan 2002, AOCS 2006).

GMO Detection Process

Several steps are necessary to detect the presence of GMO in raw materials or processed foods. The steps include: sampling, isolation and DNA extraction and purification, detection including identification of GM molecules, and quantification if necessary. Detection should done using methods that have been validated and passed proficient tests to avoid false negative or false positive results.

Sampling

The first step to detect the presence of GMO in raw materials or food products is sampling. Sampling should use accurate statistical procedures. Imagine if the sample came as shipped soybean bulk. How much soybean would be needed to be analyzed? What if the samples are thousands of canned food in a supermarket? Would we analyze each bean or each can to find one or several ones that contain GMO? For instance, 1.000 canned tomato sauces made from 10.000 non-transgenic tomatoes and 50 transgenic ones; how to detect?

Sample size and sampling procedure is the most important key in detection of raw materials or food

products containing GMO. Correct sampling will avoid the usage of heterogeneous samples. Sampling design must be carried out according to proper statistical methods, and sample quantity must be adequate to represent the materials tested. Raw materials such as seeds must be powdered and mixed homogenously, so that they can be used to extract DNA or protein. Even though we still do not have sampling methods to detect GMO, there are several institutions in some countries that have published robust sampling methods such as Grain Inspection, Packers and Stockyards Administration (GIPSA) at the Agriculture Department in the USA (USDA 2006) and the Japanese Ministry of Health, Labor and Welfare (Ministry of Health, Labor and Welfare Japan 2006), the European Union (Kay and Paoletti 2001) and Codex Alimentarius Commissions (2004).

GMO Detection Methods

Detecting GMO or its derivatives can be done normally based by DNA and/or protein from its source. The main method is to use DNA, and very few methods use protein. Protein-base methods normally are simpler, easier and cheaper to do, but have several disadvantages such as the methods can only be used with raw material samples (seeds and flour), and can not be used for sample from food that had undergone long processes such as tofu, tempe, and other food. The differences between the two methods can be seen in Table 6.

Protein-base Detection

Identification using protein needs monoclonal antibody that is produced according to the specific protein encoded by the inserted gene. These protein-base methods can be used on unprocessed materials such as seeds and half-processed materials, as long as the proteins are not yet denatured or broken due to food processing. This protein-base detection is divided into two major classes. They are Lateral Flow Strip

Table 5. Transgenic maize and its introduce genes.

Inserted gene Trade name Characteristic

Promoter Structure Terminator

Event176-maize (Novartis) Insect resistant, phosphinotricin-herbicide tolerant

1) P-PEPC p-CDPK 2) P-35S

1) two synthetic cry1A(b) 2) bar

1) I9, T-35S

Bt11-maize (Novartis) insect resistant, phosphinotricin-herbicide tolerant

1) P-35S with IVS6-int 2) P-35 with IVS2 int

1) synthetic cry1Ab 2) synthetic bar

1) nos 2) nos

MON810-maize (Monsanto) insect resistant 1) P-35S with hsp-70-int 1) synthetic cry1Ab 1) nos T25-maize (AgrEvo) Phosphinotricin-herbicide tolerant 1) P-35S 1) synthetic bar 2) T-35S GA21-maize (Monsanto) Glyphosate-herbicide tolerant 1) Pr-act with OTP 1). M-epsps 1) nos CBH-351-maize (AgrEvo) Insect resistant, phosphinotricin-

herbicide tolerant 1) P-35S 2) P-35S

1) cry9C 2) bar

1) 35S poly A signal

Source: Chiueh et al. (2002).


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(LFS) and ELISA. If LFS is used, sample must be powdered into flour, homogenized, and buffer/water is added to extract the protein, and LFS stick can be dipped into the buffer/water containing the sample. After several minutes, positive test result is indicated by colored line in the strip that is caused by reaction between antibody and the target protein. This method is the simplest and cheap to detect GMO qualitatively.

ELISA (Enzyme-Linked Immunosorbent Analysis) method also needs protein to be extracted from the test materials, which is then followed by antibody detection in the micro-well plate. Positive reaction is indicated by color reaction that can be read visually or by optical analysis (Optical Density or OD) qualitatively. To analyze qualitatively, standard curve must be made using reference materials from GMO with known concentration. To do so, one plots GMO percentage against OD results from protein reaction with antibody. Positive results produced from unknown sample can be quantified by comparing the sample OD curve with standard curve from reference material. Several researches published several the GMO detection methods of LFS and ELISA (Ahmed 2002, Brett et al. 1999, Fagan 2001, Chiueh et al. 2002) and several companies such as Strategic Diagnostics Inc. (www. sdix.com), EviroLogix Inc. (www. envirologix.com), and Neogen (www. neogen.com) have produced and sold several types of test kits to detect GMO using LFS and ELISA.

DNA-base Methods

Out of several methods that are used to detect the presence of GMO, such as PCR, LCR, fingerprint (RFLP, AFLP, and RAPD), PCR is the mostly used method and commonly accepted for meeting the requirements of regulation to obtain approval and for labeling.

DNA Extraction Methods

To date there are two DNA isolation methods that are widely used. They are CTAB method and DNA binding silica column that is normally produced as commercial test kits by foreign companies. CTAB method is based on incubation of the sample with detergent hexadecyltrimethylammonium bromide. Several DNA extraction kits based on DNA-binding silica are WizardTM produced by Promega (Wisconsin, USA) and Dneasy Plant mini kit from Qiagen (Hilden, Germany).

Efficiency of PCR, like other DNA methods, depends on the quality and purity of isolated DNA. DNA quality is measured by fragment size and the damage level. Damaged DNA can occur due to hydrolysis because of heat, low pH, and nucleases and degradation caused by other enzymes. DNA purity depends on the presence of contaminant such as polysaccharides, fat and polyphenol or other chemical substances used during isolation process. Taq polymerase activity can be inhibited by polysaccharides, EDTA, phenol and SDS (Pan 2002), so that PCR product can not be obtained.

Accurate extraction and purification methods are needed especially to remove matrix effects on food that underwent long process such as maize syrup, maize oil, soy milk, tempe, soy sauce, tofu and others.

There is a crucial aspect in GMO detection, which is the amount or quantity of GMO in samples that is very important to be identified in labeling of the product that contains GMO. The maximum GMO concentration (%) in raw materials, and processed food is the threshold in labeling regulation in some countries such as European Union, Japan, South Korea, and Taiwan. Therefore, quantitative PCR method is needed to be done.

Table 6. Similarities and differences between DNA-base and protein-base GMO detection methods.

DNA-base (PCR and equivalents) Protein-base (Lateral Flow Strip, ELISA, and equivalents)

DNA detection Protein detection Cannot be used for sample containing no DNA Cannot be used for sample containing no protein Standardization method for sampling and extraction is needed Standardization method for sampling and extraction is neededComplete well equiped laboratory is needed to obtain accurate data Simple laboratory is needed to obtain accurate data Reference material is needed Reference material is needed Very sensitive thus often results in false positive Not very sensitive Information on inserted gene is needed Information on protein expressed by inserted gene is needed Usually answers qualitative, except if competitive and real-time PCR are being used

Can give both qualitative and quantitative answers

Takes time Short time needed (1-2 hr) High cost Low cost

Sumber: www.afic.org/detecting.


Qualitative PCR

The first known PCR technique and broadly used is end-point PCR. End-point PCR is a PCR technique with results that can only be seen and examine after the PCR process finishes. Analysis is carried out by gel electrophoresis. Two primers are used in this technique. Both primers are designed to hybridize at two opposite DNA sequences in the introduced gene and then the sequence between the two sequences is amplified several million times by recycling. Amplification of the DNA fragment is then examined with gel electrophoresis that can separate DNA according to size. End-point PCR is normally used for qualitative analysis of GMO detection in food materials and food products. This process, in the detection issue, is known as screening, where the target genes are those that are commonly used in plant transformation process such as 35S and Nos. The screening can only be used to determine the presence of GMO at sample without ability to point out the specific events. To find out whether a sample contains certain events of GMO, PCR with specific primers for specific event is used. The primers usually contain part of promoter sequence and inserted gene sequence as the target sequence.

Semi-quantitative competitive PCR

Analysis with semi-quantitative competitive PCR method is usually based on comparison of final amounts of DNA amplified from two targets DNA. The first DNA is the one with amount to be determined and the second one is DNA of the competitor that is made artificially. Competitor DNA is added in small amount with known concentration before PCR process starts. This is then amplified together with target DNA to be measured. After PCR finishes, product amplification can be seen in gel electrophoresis and if the two target DNA results in same outcome, it is assumed that starting DNA amounts are also the same. With setting of two competitive PCR, one for GMO to be determined (RR soybean for instance) and one with analyzed species (non-transgenic soybean), and simultaneously adding competitors for both, GMO quantity related to the species can therefore be determined by exploring dilution and concentration level of the competitors. Some researches have published and reported that GMO detection method using competitive PCR and can showed it give accurate results (Hubner et al. 1999, Wurz et al. 1999). However, this method is still in debate because some researchers think that the only one accurate method for quantitative detection is real-time PCR. This is due

to years of experience of inconsistency with competitive PCR results.

Quantitative Real-Time PCR

Commonly used method for analysis quantitative PCR is real-time PCR. Unlike conventional PCR (end-point PCR) where visualization of amplification result can only be done after the PCR process finishes, at real-time PCR the amplification process at each cycle can be observed. PCR amplification can be analyzed using fluorescent dye or fluorescent probe. Dye that can put color in double-stranded DNA (SYBR Green I) can also be used as nonspecific system to detect amplification of all targets DNA. Specific detection is performed by fluorescent probe to recognize internal segment of target sequence, by either probe hybridization (FRET) or probe hydrolysis (TaqMan). To quantify GMO, specific detection is preferred to avoid troubles caused by nonspecific amplification. Probe use has advantages such as ability to detect and verify simultaneously target sequence. One of TaqMan probe hydrolysis techniques is to use ABI Prism 7700 that can detect 2 pg in 1 gram MM and RSS 3 hours after DNA extraction.

Several research results show that the use of real-time PCR method can detect GMO quantitatively with accurate results (Vaitilingom et al. 1999, Wurz et al. 1999). Recently, this method is the most common method used to detect GMO quantitatively, even though this method expensive and time consuming when compared to others. Besides, the method needs trained human resources.

GMO detection methods keep progressing, one of them is multiplex PCR that uses several primers in one PCR running to reduce price and time. GMO detection using microarray, capillary gel electrophoresis, biosensor and genosensor are still under studied. Those technologies are still developing stages and will need sometime for routine and economic application as an addition or a substitute of methods that are being used now.

CONCLUSION AND SUGGESTION

Conclusion

1. Globally, there was an indication of the increasing of areas planting by transgenic crops

2. Several regulations to guarantee safety of food produced from the technology also developed.

3. Indonesia already has regulations on food safety of GMO, but not yet completely implemented. One of


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the reasons was the regulation guideline is still in the making process.

4. Indonesia has four laboratories that have capability to to give service on detecting GM food, but their capacities are still need to be increased.

5. GMO detection methods based on DNA and protein are continuously growing in the world.

6. Protein-base detection method generally uses LFS and ELISA that are marketed as test kits.

7. DNA-base detection method that commonly use is PCR technique; the technique was then modified to competitive PCR and real-time PCR so that they can be used for semi quantitative and quantitative analysis.

Suggestion

Because Indonesian has been launched several regulations and policies regarding GM food safety, commitment from the government is needed to implement them in daily life. In addition, Indonesia also one of the countries which ratified the Cartagena Protocol. Therefore, to implement all regulations regarding GM food safety, the framework for implementation of this protocol should be made. Besides, the capacity of human resources and laboratories are need to be strength, so Indonesia can analyze the presence of GMO in food materials and products accurately for import and export purposes.

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