Detection of genetically modified foods

Yang Yunxia 1 Jiang Shiqiang 2 Zhao Ming 1 Dong Zhiqiang 1

Keywords: genetically modified food, genetically modified agricultural products, food safety, biotechnology, detection technology

Abstract: discuss on the question Genetically modified crops and Genetically modified food safety are discussed. The method of detection from gene and gene translation and gene express is given.
Key words: Genetically modified crops Genetically modified food food Safety biotechnology Detection technology

With the rapid development of biotechnology, genetic engineering technology has been widely used in the agricultural field. Agricultural biotechnology with genetic improvement as the core since the 1980s is setting off a second green revolution. Gene transfer can produce high-quality, high-yield, pest-resistant and stress-resistant varieties to alleviate shortages of agricultural and livestock products and food due to rapid population growth, reduced arable land, resource shortages and environmental degradation. Crisis. Taking crops as an example [1] Since the first genetically modified tomato was approved for marketing in the United States in 1994, the global GM crop planting area has expanded rapidly, from 1.7 million hectares in 1996 to 58.7 million hectares in 2002. Global sales of GM crop products surged from US$75 million in 1995 to US$2.1-2.3 billion in 1999, and are forecast to reach US$8 billion in 2005 and US$25 billion in 2010 (2).

The safety of genetically modified agricultural products and food

1. Safety thinking caused by genetically modified agricultural products and food

With the rapid increase of genetically modified agricultural products and foods, although it has many advantages such as high quality and high yield, it is because GM agricultural products and foods transfer a foreign gene fragment to the target species, thus changing the traits of the target species, especially 1998. The Pusztai incident at the Rowett Institute in Scotland, England, was later denied [3], but the incident of the Monarch Butterfly reported by Cornell University in Los Angeles in 1999 is still being further verified. The fact that Brazil nut protein and its genetically modified soybean cause similar allergic reactions to humans raises many questions about the safety of genetically modified agricultural products and foods. For example: Is the foreign gene safe? Is the genetic structure stable and harmful? Healthy mutations? Are new harmful genetic traits or unhealthy ingredients produced after gene transfer? Are nutrients and other functionalities changed? Does the genetically modified marker gene cause a drug-resistant gene? Does it increase the allergen of food [5]... Consumers consider it very natural to raise the safety of genetically modified foods. The safety of genetically modified foods has caused global controversy, and it is necessary to conduct safety testing and evaluation of the new things of genetically modified foods. Safety assessment mainly includes the safety of marker genes such as non-toxicity, non-allergic and antibiotic resistance. People all over the world are calling for a scientific assessment of genetically modified foods.

2. The principle of substantial equivalence [6]

The current assessment of food safety uses the principle of substantive equivalence proposed by the World Economic Development Cooperation (OECD) in 1993, that is, whether genetically modified foods obtained through biotechnology are substantially equivalent to current commercial foods: (1) Biotechnology Food is substantially equivalent to traditional food. Due to the long-term natural selection, the body has been well adapted to the substance, and it has been accepted by humans and the natural environment. It is not necessary to carry out safety testing and evaluation of such products. (2) Transgenic products and traditional products have substantial equivalence with non-transgenic plants and animals, except for certain specific traits inserted. The detection of such transgenic products should focus on the detection of the inserted gene expression products, without over-emphasizing the analysis of other traits, and without regard to whether the DNA and mRNA itself are toxic, because the DNA components of all organisms are the same. . (3) There is no substantial equivalence between GM products and traditional products, and there are no traditional products on the market that correspond to GM foods. This type of products requires detailed safety testing and evaluation, including the presence or absence of natural toxic substances, and the presence or absence of nutrients. And anti-nutritional factors, presence or absence of allergens, stability and insertional mutations of transgenes, pleiotropic effects and secondary effects of genes.

3. Understanding and attitudes of GM foods in countries around the world

The United States is an active advocate of GM foods. American citizens adopt a more tolerant attitude toward GM foods, and the sown area of ​​GM crops has always occupied the first place in the world. They believe that GM foods undergo strict market access testing before they go on the market, and they can be marketed in virtually no difference from traditional foods. The United States is responsible for the evaluation, testing and monitoring of genetically modified foods by the FDA, EPA, and USDA. These institutions are consistent with the principles of new food management in the United Nations Food Organization (FAD), the World Health Organization (WHO), and the National Academy of Sciences (NAS) [7].

The EU is cautious about GM foods, and the government is opposed to the influx of GM food into its own country. They believe that genetically modified foods insert a heterogeneous gene from other animals, plants and microorganisms into the target food through biotechnology. Breaking the boundaries between biological species will destroy the natural food composition system, causing genetic level disorder, which may lead to some inheritance. Unexpected changes in learning or nutrients that endanger people's health. At the same time, it is also believed that the new combinations and traits of GM foods are expressed in different genetic backgrounds. People still lack their understanding of the environment and human influences. In addition, the time spent on production and consumption is still very short, and there is not enough evidence to prove the transgenics. Food is completely safe to the human body. But there are also political resistance to trade barriers. The food labeling regulations established by the European Union in 1996 stipulate that GM foods must be labelled. Management is divided into horizontal technology-related regulations and vertical product-related regulations [8].

Some countries in the Middle East are more excluded from GM foods due to the influence of religion. South America, Japan, South Korea and other relatively neutral countries do not recognize GM foods as much as the EU and do not recognize them as the United States. They have started experimental research on the safety of genetically modified foods. China not only actively supports the research of transgenic technology, but also adopts a cautious attitude towards GM foods, and recognizes the principle of substantive equivalence of GM foods that are popular in the world. The National Science and Technology Commission of China promulgated the "Genetic Safety Measures" in 1993. In 1996, the Ministry of Agriculture officially implemented the "Regulations on the Safety Management of Agricultural Biological Genetic Engineering", from the experimental research, intermediate tests, environmental release to the final commercialization of genetically modified agricultural products. The process carries out safety evaluation and has established corresponding supervision and testing measures. In March 2002, the “Administrative Measures for the Safety Evaluation of Agricultural Genetically Modified Organisms” and the “Administrative Measures for the Identification of Agricultural Genetically Modified Organisms” were formally implemented. The promulgation and implementation of these policies and regulations provide a strong guarantee for people to eat genetically modified foods. 〔9〕

Method for detecting genetically modified food

In view of the worldwide concern and fierce debate about the safety of genetically modified foods, the detection of genetically modified foods is particularly important. The genetic engineering technique is to transfer the cloned foreign gene into a target crop by a method such as a gene gun method or an Agrobacterium-mediated method, and to have a superior trait of a foreign gene in the target crop. Detection of genetically modified foods, analysis from the transgenic technology itself, can be carried out at the genetic level, gene transcription levels, and gene translation product levels.

Gene level detection

When transgenic technology inserts a gene fragment of interest cloned from various organisms into a targeting receptor, it is generally necessary to construct a promoter, a terminator, a selectable marker gene, a reporter gene, and the like. The detection of the transgene at the gene level is to detect the nucleic acid sequence of the receptor, the integration site of the target fragment, the gene polymorphism, the content analysis of the target fragment, and the promoter, terminator, selectable marker gene, reporter gene, and the like.

Polymerase chain reaction PCR method [10]

The PCR sample is applied to the sample to be tested, and suitable primers are designed for the foreign gene, and the target gene fragment is rapidly and specifically amplified by the polymerization of Taq DNA enzyme, so that the target gene fragment can be amplified in an order of magnitude in vitro and can be obtained in a short period of time. Amplify the specific foreign DNA fragment of Pg level to ng or even ug level within a few hours, and the amplified product is subjected to agarose gel electrophoresis or polypropylene gellane gel electrophoresis, stained with ethidium bromide (EB), in the ultraviolet Exogenous target fragments can be observed under light.

(1) Qualitative PCR

Qualitative PCR can directly detect promoters, terminators, marker gene fragments, and target gene fragments. In order to allow the expression of the gene to be well expressed, a promoter and a terminator are often added to the 5' and 3' ends of the gene of interest, respectively, when constructing the gene expression vector. Currently, approximately 75% of transgenic plants use the Ca MV (Cauliflower mosaicvirus) 3 5 S promoter, followed by the NOS promoter of the nopaline and octopine synthase and the Ocs promoter. A commonly used terminator is the NOS terminator of the nopaline synthase and the 3' end region of the Rubisco small subunit gene, so the current detection of promoters and terminators is actually the detection of the Ca MV35S promoter and the NOS terminator. However, some plants such as cruciferous plants are susceptible to CaMV infection and carry 35S, so a positive signal is detected; while NOS terminator is derived from the ubiquitous Agrobacterium tume faciens, therefore, the 35S promoter and NOS are terminated by PCR. Other tests should be used in conjunction with the test. Qualitative PCR is affected by DNA extraction and purification. If the DNA is degraded or contaminated, the test results will be false positive; only GMF can be determined initially but it is not possible to identify which GMF.

(2) Quantitative PCR

In actual trade and food consumption, we not only want to know whether the food is genetically modified, but also want to know the content of the foreign gene, that is, the percentage of the foreign gene in the GMF. In the normal qualitative PCR amplification process, the positive samples with different concentrations are used as reference, the standard positives are synthesized by genetic engineering methods, the fluorescent markers are referenced upstream, the downstream primers are not labeled, and the standard positives are also amplified while the template is being amplified. Amplification. . Finally, a standard curve of the absorbance and the transgenic content is made based on the absorbance value, so that the transgenic content of the test sample can be determined, so that semi-quantitative detection can be performed.

(3) Quantitative competitive PCR (Quantitative competitive PCR)

In the reaction tube of the PCR, the template labeled with the fluorescent label for artificial synthesis and the sample to be tested are simultaneously placed, and both are simultaneously amplified by the same primer. After the reaction is completed, the DNA content of the sample to be tested is estimated by measuring the fluorescence intensity.

(4) Real-time quantitative fluorescence PCR method [11]

The real-time quantitative fluorescent PCR technique is based on conventional PCR and adds a fluorescently labeled gene probe (generally Taq man). One is labeled at the 5' end of the probe, and one is labeled at the 3' end of the probe. The two form an energy transfer structure. The two fluorescent groups are absorbed or suppressed according to the distance control, and the reaction system is continuously detected in the continuous amplification of the PCR. The change in fluorescence signal determines the amount of starting DNA by linearly recording the number of cycles and the logarithm of the amount of starting DNA in the PCR system. Detection of genetically modified foods by real-time quantitative fluorescent PCR can avoid false positive results caused by external pollution or DNA degradation of the sample during ordinary PCR reaction, but the real-time quantitative fluorescence PCR instrument is expensive and the detection cost is high.

(5) Reverse transcription RT-PCR

Reverse transcription PCR overcomes the special amount of exogenous gene in the sample to be tested, and reverse transcribes into cDNA using mRNA as a template, and then detects by PCR amplification.

(6) PCR-ELISA

PCR-ELISA is a transgenic detection method that combines the high efficiency of PCR with the high specificity of ELISA. The oligonucleotides covalently cross-linked on the PCR tube wall are used as solid phase primers, and under the action of Taq enzyme, the target nucleic acid is used as a template for amplification, and a part of the product is cross-linked on the tube wall, which is a solid phase product. Free in liquid, liquid product. For the solid phase product, the labeled probe can be used to hybridize with it, and then alkaline phosphatase-labeled streptavidin is used for ELISA detection, and the liquid phase product can be analyzed by gel electrophoresis. Combining the two methods of PCR and ELISA can complement each other and make the detection accuracy greatly improved. At present, there is no unified screening method for genetically modified foods in China. With the increase in the number of genetically modified foods and China's accession to the World Trade Organization, the establishment of genetically modified food screening methods in China will play a positive role in the protection of population health and ecological environment.

Southern hybridization

Southern hybridization techniques are used in molecular biology for specific sequence localization of genomic DNA. Southern technology in GM foods is used in cases where the GM food is known to be transferred to a foreign gene fragment. A homologous sequence of a radioactively or fluorescently labeled exogenous gene of interest is used as a probe to hybridize with the total DNA of the food product agricultural product. The receptor total DNA is first digested with restriction enzymes, and the resulting fragment is separated by size by agarose gel electrophoresis, and then the DNA is denatured in situ and transferred from the gel to a solid support. During the transfer of DNA to the solid support, the relative positions of the individual DNA fragments remain unchanged. The radioactive or fluorescently labeled probes are used to hybridize with the individual DNA fragments, and the position of the electrophoresis strip complementary to the probe is determined by autoradiography. . Southern technology is used for the detection of foreign genes in foods to detect DNA fragments with high homology between the foreign gene and the endogenous gene, and is accurate and reliable, but the purity of the sample is higher and the cost is higher.

Gene chip

The gene chip [12] is a high-tech developed rapidly in the field of life sciences in the late 1980s. It is a matrix densely arranged by a large number of DNA or oligonucleotide probes. The gene chip can use the current common reporter gene. a specific fragment (target fragment) of the resistance gene, promoter and terminator is immobilized on a slide to prepare a detection chip, and the DNA extracted from the sample to be tested is amplified, labeled, and hybridized with the chip, and the hybridization signal is scanned by the chip. Instrument detection, and then through computer software for analysis and judgment, the basic principle is to detect signals by hybridization. Gene chip technology allows for intensive and parallel processing of samples. The use of gene chips enables accurate, fast, and large-volume detection of DNA. Therefore, the sensitive and accurate detection of a large number of transgenic crops to be tested is conveniently and quickly.

The downside is that each test sample must first have a matching non-GM crop chip.

2. Gene transcription level detection

Northern blot hybridization

The use of northern technology for the determination of foreign genes can determine the size and abundance of transcript mRNA molecules of a particular foreign gene DNA. The RNA molecules are separated from each other in a denatured agarose gel according to their size, and then the RNA is transferred to an activated cellulose, nitrocellulose filter. The transcript RNA of the foreign DNA is determined by hybridization and autoradiography with a radiolabeled exogenous DNA probe or RNA probe.

3. Detection of gene translation expression levels

1. Enzyme-linked immunosorbent assay (EL ISA) technology

Enzyme-linked immunosorbent assay (E nzy me-Linked Immunosorbent Assay, EL ISA) is used to detect the expressed protein of interest. The basic principle is the specific binding of antigen and antibody and the efficient catalysis of the enzyme on the substrate. The antibody against the protein of interest is coated on the ELISA plate, and the solution of the analyte is added. After incubation, the expression product of the transgenic fragment, ie, the corresponding protein, is contained in the sample to be tested, and the protein is used as an antigen and a coated specific antibody. Combine. The enzyme-labeled antibody against the target protein was added, and after incubation, the enzyme reaction substrate was added to develop color, and the OD value was measured by an enzyme-linked reader.

2. Protein chip

The operation of the protein chip is the same as that of the gene chip, except that the principle is to utilize the specific binding of the antigen-antibody rather than the complementary hybridization of the base pair.

3. Westertern blotting

The principle of Western blotting is similar to that of Southern hybridization and northern blot hybridization, and is a specific binding of antigen-antibodies. The protein extracted from the sample is separated into molecules of different sizes by gel electrophoresis, and transferred to a solid support, hybridized with a radiolabeled antibody as a probe, and detected by radioactive autoradiography. The expression product of the gene. Western blotting and EL ISA are both specific binding of antigen and antibody, and the labeled probes are different. One is a radioactive label and the other is an enzyme label.

Development direction and trend of genetically modified food testing

Since genetically modified foods are directly related to people's health and vital interests, the detection of genetically modified foods is fast, accurate, practical, and easy to operate. The detection of genetically modified foods is moving in the direction of kits, and the role of DNA chip technology in future transgenic detection is becoming more and more prominent.


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