Screening of Exist Genetically Modified Elements in Local and Commercial Rice Available in Baghdad Markets Using PCR and Real-time PCR

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INTRODUCTION
Rice has been considered as staple foods consumed by Human beings, where more than half of the world population used rice as a daily essential food, especially in Asia and the third world countries such as Iraq (Kameswara and Jackson, 1997;Londo et al., 2006).Rice has been chosen as a model organism for the molecular biology research because of the economic importance and the small size of its genome (just 430Mb), where it was the first crop that its genome sequence was completed (Bennetzen, 2002;Chao et al., 2003).
The rice cultivation faced many problems such as shortage of water resources or drought; lower yield; diseases and insects' infections and other problems (Zhang, 2007).All these0020problems can be solved by improving agricultural practices; using traditional plant breeding techniques or modifying the genetic code using modern scientific technologies, these technologies those known as gene technology or genetic modification technology capable of overcoming the problems more quickly than traditional methods (Verma et al., 2011).
The scientists developed a rice crop using genetic modification technology, where they include many traits to rice such as quality improvement; yield enhancement, drought tolerance and insects and diseases resistance as described in the following studies: Paine et al., (2005), Bandyopadhyay et al. (2007), Kanneganti and Gupta (2008), Tang et al. (2006) and Datta et al. (2002) respectively.The products of these studies known as genetically modified foods (GM Foods) or Genetically Modified Rice (GM Rice).
Genetically modified food is a term referring to genetically modified plants or/and the food contains components of genetically modified plants, as well as products of genetically modified organisms and this food uses for human and animal consumption (Whitman, 2000;Verma et al., 2011).GM Food is one of the best methods that have solved many problems such as nutritional values, world hunger, environmental contamination and other global issues.Since 1996, the cultivation of genetically modified crops has been growing; which, as a result, led to a need of global legislation to regulate the presence of genetic modification in food, rate of genetic modification, labeling for GM foods and methods of detection (James, 2003;Rizzi et al., 2004).
The most common methods of GMOs detection are Enzyme Linked Immunosorbant Assay (ELISA) and Polymerase Chain Reaction (PCR), but the best method is PCR because it is more specific and sensitive, despite being more expensive, time consuming and cannot be done on site (Brandner, 2002;Jasbeer et al., 2008;Vidhya et al., 2012).There are many types of PCR such as conventional PCR, which is used for GMOs detection (qualitative) and Real-Time PCR, which is used for GMOs detection and rate of genetic modification determination (qualitative and quantitative) (Rizzi et al., 2004;Abdel-Mawgood et al., 2010).Moreover, Real-Time PCR is preferred in routine work because it shorter time if compared to conventional PCR that needs gel electrophoresis to analyze the results (Vidhya et al., 2012).
The aim of this study is determined if there is any GM rice in Baghdad markets using conventional PCR and Real-Time PCR.

Sampling and genomic DNA extraction:
The samples of rice seeds that are cultivated in Iraq collected from the State Board for Agricultural Research (SBAR) and AL-Mashkhab Rice Research Station (MRRS) while the samples of commercial rice seeds collected randomly from local market in Baghdad during the period between January-May, 2013.Table 1 shows the details of 42 rice samples including 7 samples of rice seeds that cultivated in Iraq; 31 samples of commercial rice seeds and 4 samples of kids' food, where rice is one of their ingredients.Standard protocol (The isolation of genomic DNA from plant tissue protocol) of wizard genomic kit (DNA purification kit, Promega, USA) was used for DNA extraction from 60 mg of ground seeds.The quality and quantity of extracted DNA was determined by using ActGene micro-volume UV/Vis spectrophotometer (Avans, Taiwan).

Real-time PCR (reactions and program):
The Real-Time PCR reaction was performed using AccuPower GreenStar qPCR PreMix Kit (Bioneer-Korea), this premix pellet containing all required components for qPCR reaction with the exception of the DNA template and primers.Furthermore, this pre-mix containing SYBR Green dyes for monitoring the amplification process.
After that, the final volume was adjusted to 20 µL with DEPC-distilled water.The Real-Time PCR program conditions for GMOs detection were carried out, initial denaturation, at 95°C for 5 min (1 cycle), followed by 45 cycles of denaturation: at 95°C for 30 Sec, annealing: at 60°C for 30 Sec, extension: at 70°C for 30 Sec and scan the fluorescent of SYBR Green dye.Subsequently, the PCR products exposed to melt by increasing 1°C every 2 Sec starting from 60 up to 95°C to make sure the products specificity.All PCR runs, conventional PCR and Real-Time PCR, included negative and positive control samples.To detect any contamination, negative control reaction was set in each PCR experiment, a negative control reaction containing all components of the reaction without DNA template.On the other hand, a positive control was prepared to determine the effectiveness of the conditions of PCR reaction and program.A positive control reaction containing all components of the reaction with DNA template of standard sample, which containing the target regions (RM 171 and/or CaMV35S promoter).

Agarose gel electrophoresis analysis:
The PCR products and 100 bp DNA ladder bands (Promega, USA) were separated using 2% agarose gel electrophoresis, then stained the gel with ethidium bromide and visualized under the ultraviolet light (302nm) (Sambrook and Russell, 2001).The expected molecular size of PCR products were about 305-331 bp for primer RM171 and about 101-110 bp for primer P35S.

RESULTS AND DISCUSSION
To detect if there is any GM rice grain in Baghdad markets, 42 samples were collected from different regions of Baghdad; all collected samples were known origin and certified.DNA extracted using DNA purification kit (Promega, USA); this commercial kit was suitable method for DNA extraction from dry seeds comparing with the traditional methods.Moreover, this method is not time-consuming as traditional methods.DNA yield and purity ranged between (435.5-1141ng/µL) and (1.32-1.98),respectively.
The PCR results of RM171 detection used to confirm that the studied samples were rice and/or the rice was one of food ingredients, as well as to confirm the DNA integrity for being used in PCR experiments.All tested samples show positive results (band with size 330 bp), for RM171marker except sample no.(38) (Fig. 1) because this sample was the food ingredients of Kids against Hunger food that prepared from the following components: Textured Vegetable; Soy; Modified Corn Starch and other materials.That means all samples contain rice genome and the extracted DNA was apt for using in PCR experiments.This result is similar to those of previously published studies those used different species specific genes such as lectin and zein genes to detect species of soybean and maize, respectively (Al-Rousan et al., 2010;Yoke-Kqueen et al., 2011), also to confirm the DNA integrity to be used in PCR experiments.The 35S promoter and the NOS terminator exist in most of the transgenic crops and these elements known as the GM elements (Abdel-Mawgood et al., 2010;Alaraidh et al., 2011;Vidhya et al., 2012).In this study, the PCR experiments were performed using primers (P35S) to detect the presence of GM element (P35S)  38) gave the highest peak in the amplification-curve as an indicator of exist the GM element (P35S) comparing with the other samples as shown in Fig. 3A, this peak almost similar to the transgenic standards (Positive control samples (Pc)) peak.
Furthermore, the cycle threshold value (Ct) was determined to detect the amplification efficiency.The average Ct value of sample no.(38) was 30.63 compare with the average Ct value of transgenic standards, or positive control (Pc) was 23.96 while average Ct value was undetermined for the other studied samples and negative control sample (Nc) that indicated the absence of GM elements (P35S) in their DNA.Two replicates were performed for each sample with each run as evident from Table 3.
Figure 3B shows the melt-curve to the products specificity because SYBR Green Dye may bind to double strand DNA such as specific/non-specific PCR products or primer-dimmers (Vidhya et al., 2012).If the PCR products were specified, they would have the same length; thus the melt peaks of these products were at the same temperature while the non-specific PCR products have different temperature depending on the product's length and other factors.These results came to confirm the results of conventional PCR and to detect the SYBR green kit effectiveness for using in the future in the routine work for detecting GM Food.This method is a rapid process for food testing, where it gives a reliable result within a few hours without the need for agarose gel electrophoresis analysis (Rizzi et al., 2004).
As expected, all Iraqi studied samples were not GM Crops because these samples are local and ancient Iraqi varieties and there was no ability to produce the GM Crops in Iraq.The common indicator of GMOs (P35S) was not detected in the rest of the samples studied, some of these samples certified and labeled from their sources as products that are not genetically modified.Sample no. ( 38) is food from the organization of Kids Against Hunger as a food aids for kids in developing countries.This kind of food has been introduced to Iraq without any GM food detection, although there is decision no.128 that issued from the Advisory Board of the Food in July 2002 ban the import; introduce; use and transfer of GM foods to Iraq (Aarif et al., 2013).
When making contact with the Kids against Hunger organization if there is any permission for using GM crops by the authorized agencies such as United States Department of Agriculture (USDA) and if there was permission, why their products did not label from these responsible agencies?The answer was as following: "We are working on that as we speak.It is very involved process to get them to approve anything.We are hoping to have this done by the end of the summer!" (Private connection by e-mail).That means there is no allowance till now to marketing this product.
In these days, there are no rules for regulating the GM food introduction and their products into Iraq; thus, it is easy to enter such foods.This study is not the first in GM food detection in Iraq, but it is the first in GM rice detection in Iraq.There are a number of GM food detection researches with other types of food, where Aarif et al. (2013) study indicated that there are GM Corn (Zea mays) in the Iraqi markets while Hassan and Ali (2012) study observed presence of the transgenic sequences of maize in three samples of milk that collected from the Sulaimani market in Iraq.

CONCLUSION
In this study, genetically modified elements were detected in rice samples using PCR and Real-Time PCR.The PCR techniques used in the GM food detection has been sensitive and effective.All the Iraqi rice varieties were not GM crops that mean there was no ability to produce any GM crops in Iraq.Also, we can conclude that the monitoring of the GM food entry to Iraq is limitable and there is no commitment neither in labeling some foods that genetically modified, nor in taking approvals from responsible organizations or agencies.

Table 1 :
The details of rice samples and the PCR results PCR results

Table 2 :
The PCR programs conditions Time and temperature

Table 3 :
The cycle threshold value (Ct) of 35S promoter for samples (31 to 42); positive control (Pc) and negative control (Nc) only one out of 42 tested samples gave a result (Fig.2).GMO element detected with sample no.(38); this sample contains modified corn starch as one of the food ingredients; therefore, this result was expected.Real-Time PCR experiments have shown the same results of conventional PCR.Sample no. ( and