Method for detecting Bt gene in transgenic rice

In July 2014, CCTV reported on the news of the presence of genetically modified rice in Hunan, Hubei, Anhui, and Fujian provinces, which caused great concern in society. The reporter randomly purchased 5 kinds of rice in a large supermarket in Wuhan. After testing, it was found that three of the five rice varieties contained the genetically modified component: Bt63. Bt63 is an insect-resistant gene, which is an insecticidal crystal protein gene in the Bacillus thuringiensis gene, which can effectively prevent a variety of harmful insects such as lepidoptera.

In recent years, genetically modified events have emerged in an endless stream. So what is a genetic modification? A transgenic technique is a process of extracting a gene of interest required in a genome of a specific organism or artificially synthesizing a DNA fragment of a specified sequence, transferring the gene fragment into a specific organism, and recombining with its own genome to obtain an individual having a stable expression-specific genetic trait. . Crop transgenics are usually used to obtain antiviral, insect resistance, cold resistance, drought resistance, sputum resistance, salt and alkali resistance, herbicide resistance and other characteristics.

China does not currently approve commercial production of genetically modified rice. European Commission Resolution: Chinese rice products must enter the EU market with a certificate that does not contain genetically modified Bt63 rice. The explanation is: “The GM crops can only be allowed to enter the European market after comprehensive scientific evaluation and approval.” The decision was made to prevent unapproved GM Bt63 rice from being purchased and consumed by European consumers. It can be seen that the identification of Bt genes in rice is critical. There are many methods for detecting Bt gene in rice. RephiLe launched the rice Bt gene detection solution based on the Ministry of Agriculture 953 Announcement-6-2007_Transgenic plants and their product components for the detection of insect-resistant Bt rice qualitative PCR method. ,details as follows:

First, the experimental principle

Primers were designed based on the specific sequence of the transgenic rice, and the samples were subjected to PCR amplification. The PCR results are tested to determine if they contain a transgenic fragment.

Second, instruments and reagents

PCR instrument MyCycler (biorad, USA)

Benchtop micro-refrigerated centrifuge Microfuge 22R (BeckmanCounter, USA)

Gel Imaging System Gel DocTM XR (biorad, USA)

UV detector NanoDrop1000 (eppendorf, German)

Nucleic Acid Electrophoresis System EPS100 (Shanghai Tianneng)

Ultra-Pure Water Direct-Pure UP (RephiLe)

Constant temperature water bath HH.S.21.8 (Shanghai Yuejin Medical Instrument Factory)

Third, the test method

1, sample pretreatment

The rice is pulverized to prepare a sample to be tested.

2, genome extraction

The Axygen genomic DNA mini kit was used and extracted according to the method described therein, and the content was detected by UV.

3, primer design

4, PCR conditions

Reaction system: Using DBI 2xTaq PCR Master Mix, template and primers were added as indicated.

Reaction conditions: first stage 95 °C denaturation 5 min; second stage 35 cycles: 94 °C denaturation 1 min, 56 °C annealing 30 s, 72 °C extension 30 s); third stage 72 °C extension 7 min.

5, nucleic acid detection

The PCR product was subjected to gel electrophoresis. The SPS amplification product was 277 bp in size and the Bt amplification product was 301 bp in size.

6, the result is confirmed

Using the plasmid containing the Bt gene as a positive control, it was confirmed that the rice containing no Bt gene was used as an imprinted control. The positive control SPS and Bt genes were amplified, and the negative control only amplified the SPS gene.

If the sample to be tested amplifies the SPS gene, it indicates that it contains rice components.

If the SPS gene and the Bt gene are amplified, it indicates that the transgenic rice component is contained.

Ordinary PCR experiments can only determine if a sample contains a genetically modified component. Need to confirm the GM rice content can be tested by Realtime PCR.

PCR experiments require very high levels of water for experiments, and contaminants in water can cause different damage: metal ions, especially Mg, can affect the activity of Taq enzymes. Organic matter, especially humic acid and fulvic acid, associate with DNA or interfere with enzymes and affect fluorescent signals; DNA enzymes degrade DNA. Bacteria release ions and organic matter during metabolism, causing various disturbances. Therefore, ultra-pure water with no ions, no organic matter, and no bacteria must be used in the PCR experiment.

In order to ensure the accuracy of PCR experiments, life science laboratories are equipped with high-quality ultra-pure water meters for experiments. For example, the Shanghai Institute of Nutrition, Chinese Academy of Sciences, Yuan Longping Laboratory of Hunan Agricultural University and other well-known biological laboratories use RephiLe Direct. -Pure UP ultra pure water meter. The Direct-Pure UP ultra-pure water meter uses tap water as the influent water to produce RO pure water and ultrapure water. It uses two sets of large-capacity double-tube purification columns to effectively remove trace metal ions in water, and the water production resistivity can reach 18.2 MΩ•cm. The internal ultra-pure water timing cycle, combined with dual-wavelength UV lamps, can reduce organic levels in water to below 5 ppb. The terminal is specially equipped with ultrafiltration to remove the pyrogen column, which can effectively remove biological macromolecules such as bacteria and pyrogens. The Direct-Pure UP Ultrapure Water Meter is suitable for use in molecular biology experiments involving PCR. RO pure water can be used to wash experimental vessels, ultrapure water can be configured with electrophoresis Buffer, blank control, etc.

Genetic modification is a serious science. The public's trust in the safety of genetically modified foods is very low, and it is relatively high for non-food crops such as genetically modified cotton. All experimental data at this stage does not prove that GM foods have an impact on the health of consumers. Of course, the time for the introduction of the GM is not long, the number of experimental samples is still low, and the long-term results cannot be verified. Life science is a very broad science that may not be exhausted for a lifetime. We support GM trials, which can greatly help alleviate the growing demand for food; however, commercial GM foods need to be freely chosen by the public, GM foods must be clearly labeled, and everyone has the right to choose. With the deepening of GM research, one day, safe GM foods will enter thousands of households and contribute to the development of the world.