CN101724699B - Method and special kit for testing whether rice to be tested is transgenic rice 'Kefeng No.6' - Google Patents

Abstract

The invention discloses a method for detecting whether the rice to be tested is the transgenic rice Kefeng No. 6 and a special kit thereof. The invention provides a specific primer pair and a specific probe for the boundary sequence of the exogenous insertion fragment of the transgenic rice line Kefeng No. 6. The specific primer pair is a primer pair composed of nucleotides shown in sequence 1 of the sequence listing and nucleotides shown in sequence 2 of the sequence listing. The nucleotide sequence of the specific probe is shown in sequence 3 of the sequence listing. The method provided by the invention uses the total DNA of the rice to be tested as a template, and uses the specific primer and the specific probe to perform real-time fluorescent PCR detection to determine whether the rice to be tested is the transgenic rice line Kefeng No. 6. The kit provided by the invention includes the specific primer pair and the specific probe. The specific primer pair, specific probe, kit and method provided by the invention can quickly detect whether the rice to be tested is the transgenic rice line Kefeng No. 6, providing a good guarantee for the safety of the transgene.

Description

Method for detecting whether rice to be tested is Kefeng 6 transgenic rice and special kit thereof

technical field

The invention relates to a method for detecting whether the rice to be tested is Kefeng 6 transgenic rice and a special kit thereof.

Background technique

Rice is one of the most important food crops in my country and even in the world. It is the crop with the largest population and the longest history in the world. With the development of biotechnology, transgenic technology is being widely used to improve the quality of crops. Several transgenic rice strains have been released in the environment and applied for commercial planting. Kefeng 6 is a transgenic rice line with a bivalent insect-resistant gene, and the foreign gene introduced is Bt/CpTI. Strain-specific detection of transgenic plants is an important technical basis for the supervision and management of transgenic plants to ensure their healthy development. The border sequence of the exogenous insert in a transgenic plant is one of the most important molecular characteristics of a transgenic plant line, which represents a transgenic event, a line. Therefore, the detection of border sequences, that is, strain specificity, is an important means to ensure the safety of transgenes.

In today's society where the safety of genetically modified organisms has received more and more attention, biotechnology-based genetically modified detection methods have become an effective means of supervising genetically modified plants and food. Real-time fluorescent PCR (Real time PCR) is one of the most commonly used and most effective methods. Real-time fluorescent PCR is based on the addition of fluorescent labeled probes on the basis of conventional PCR, through the real-time detection of the fluorescence signal of each cycle product in the PCR amplification reaction, the quantitative and qualitative analysis of the initial template is realized, which has high specificity and high sensitivity. advantage.

Contents of the invention

The purpose of the present invention is to provide a method for detecting whether the rice to be tested is the transgenic rice Kefeng No. 6 (transgenic rice line Kefeng No. 6) and a special kit thereof.

The invention provides a specific primer pair and a specific probe for the boundary sequence of the exogenous insert segment of the transgenic rice line Kefeng No. 6.

The specific primer pair is a primer pair composed of the nucleotides shown in sequence 1 of the sequence listing and the nucleotides shown in sequence 2 of the sequence listing.

Specific primer pairs:

Upstream primer: 5'-ACGTAGTACGTACCGCCGTG-3'; sequence 1 of the sequence listing;

Downstream primer: 5'-AGTGCAGATGCATGAATCGC-3'; sequence 2 of the sequence listing.

The specific probe can be a TaqMan fluorescent probe, and its nucleotide sequence is shown in sequence 3 of the sequence list. TaqMan fluorescent probe is an oligonucleotide probe, the reporter fluorescent group is attached to the 5' end of the probe, and the quencher fluorescent group is attached to the 3' end of the probe. During PCR amplification, a specific fluorescent probe is added while adding a pair of primers. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group; during PCR amplification, the 5′- The 3′ exonuclease activity degrades the probe by enzymatic digestion, so that the reporter fluorescent group and the quencher fluorescent group are separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, every time a DNA strand is amplified, a fluorescent molecule is formed , realizing the complete synchronization of the accumulation of fluorescent signals and the formation of PCR products.

The 5' end of the specific probe is labeled with a reporter fluorescent group, and the 3' end is labeled with a quencher fluorescent group. The reporter fluorescent group can be Fam (FAM), Hex (HEX), Tet (TET), Joe (JOE), Vic (VIC), Fite (FITE), Cy3 (CY3) or Cy5 (CY5). The quenching fluorophore can be Tamra (TAMRA), Rox (ROX), Dabcy (DABCY), Bhq1 (BHQ1 ) or Bhq2 (BHQ2). Nucleotide sequence of the specific probe: 5'-CCGCGCGTTGTACTGAGAACCA-3'. In the specific probe, the reporter fluorescent group may specifically be a FAM fluorescent group, and the quenching fluorescent group may specifically be a TAMRA fluorescent group.

The specific primer pair and/or the specific probe can be applied to prepare a kit for detecting whether the rice to be tested is the transgenic rice line Kefeng No. 6.

The invention also protects a kit for detecting whether the rice to be tested is the transgenic rice line Kefeng No. 6, which includes the specific primer pair and/or the specific probe.

The kit can be applied to detect whether the rice to be tested is the transgenic rice line Kefeng No. 6.

The invention simultaneously protects a method for detecting whether the rice to be tested is the transgenic rice line Kefeng No. 6, which uses the genomic DNA (total DNA) of the rice to be tested as a template, and is characterized in that: the method uses the specificity Real-time fluorescent PCR detection is performed on the primer pair and the specific probe to determine whether the rice to be tested is the transgenic rice line Kefeng No. 6.

In the PCR system of the real-time fluorescent PCR, when the reaction starts, the concentration of the two primers in the specific primer pair can be 0.2 μmol/L, and the concentration of the specific probe can be 0.1 μmol/L .

The parameters of the real-time fluorescent PCR are specifically as follows: 50°C, 2min; 95°C, 10min; 95°C, 15s, 60°C, 1min, a total of 40 cycles.

The specific primer pair and specific probe provided by the invention are designed according to the border sequence of the exogenous insert fragment of the transgenic rice line Kefeng No. 6, which has good detection specificity and high sensitivity for detecting real-time fluorescent PCR. The detection method provided by the invention has high accuracy, sensitive response and short required time. The kit provided by the invention is very convenient to use. The specific primer pair, specific probe, kit and method provided by the invention can quickly detect whether the rice to be tested is the transgenic rice line Kefeng No. 6, providing a good guarantee for the safety of the transgene.

Description of drawings

Figure 1 shows the strain-specific results of real-time fluorescent PCR detection; 1: Kefeng 6.

Fig. 2 is the sensitivity experiment result of the detection method of the present invention.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Genetically modified rice line Kefeng 6 (GMO+) (transgenic CpTI rice): Chinese Academy of Inspection and Quarantine; Rong Jun, Song Zhiping, Su Jun, Xia Hui, Wang Feng, Lu Baorong. Bt/CpTI transgenic rice and its non-transgenic Transgene drift of parental controls under spaced planting conditions. Biodiversity, 2006, 14(4): 309-314. Kefeng 6 was provided by Wang Weixia of China Rice Research Institute, and was jointly cultivated by Wang Feng, Institute of Biotechnology, Fujian Academy of Agricultural Sciences, and Zhu Zhen, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences in 1994.

Transgenic rice strain Bt63: Chinese Academy of Inspection and Quarantine; Zhao Weidong, Zheng Wenjie, He Yan. Qualitative and quantitative detection of BT63 transgenic rice by fluorescent PCR [J]. Food Research and Development, 2009, (03): 133-135.

Non-transgenic rice line Minghui 86 (GMO-): Chinese Academy of Inspection and Quarantine; Zhang Jianxin, Zheng Jiatuan, Xie Huaan, Luo Jiami, Huang Xianbo, Deng Zeqin, Wu Lanying. Breeding of new rice germplasm Minghui 86 and its serial combinations[ J]. Plant Genetic Resources Science, 2001, (01): 32-36. .

Transgenic tomato line Huafan No. 1: China Academy of Inspection and Quarantine; Huang Wensheng, Chen Hongyun, Zhao Wenjun, et al. Strain-specific detection method of transgenic extended-ripening tomato "Hua Fan No. 1" [J]. Phytosanitary, 2005, 19 (6): 321-325.

Zhongmian 41, a transgenic cotton line: China Academy of Inspection and Quarantine; Liu Shengrong, Xia Zhiming, Liu Dangpei. Research on the High Yield, Stable Yield and Simplified Cultivation Techniques of Bivalent Insect-Resistant Cotton Zhongmian 41[J]. China Agricultural Science Bulletin, 2005 , (03): 166-168.

Transgenic maize line BVLA430101 with phytase gene: Chinese Academy of Inspection and Quarantine; Rumei Chen, Guangxing Xue, Yunliu Fan et al., Transgenic maize plants expressing a fungalphytase gene. Transgenic Res (2008) 17: 633-643.

Embodiment 1, the preparation of kit

Design and screen specific primer pairs and specific probes based on the border sequence of the exogenous insert in Kefeng No. 6 for the preparation of kits.

The kit consists of the following specific primer pairs and specific probes (TaqMan probes).

Specific primer pairs:

RB-F (upstream primer): 5'-ACGTAGTACGTACCGCCGTG-3';

RB-R (downstream primer): 5'-AGTGCAGATGCATGAATCGC-3'.

The 5' end of the specific probe is labeled with the reporter fluorescent group FAM, and the 3' end is labeled with the quencher fluorescent group TAMRA. The nucleotide sequence is: 5'-CCGCGCGTTGTACTGAGAACCA-3'.

Embodiment 2, the specific detection of kit

The kit prepared in Example 1 was used to detect Kefeng 6, Minghui 86, Huafan 1, Bt63, Zhongmian 41 and BVLA430101 respectively to verify the specificity of the kit. The same detection method was used for each sample, including two steps of total DNA extraction and real-time fluorescent PCR.

1. Extraction of total DNA from the sample

1. Take about 0.1g of sample leaves in a mortar, add liquid nitrogen and quickly grind to powder.

2. Quickly transfer the leaf powder into a 2ml centrifuge tube with 700μl CTAB buffer in advance, mix gently, and keep warm in a 65°C water bath for 30 minutes. Shake carefully every ten minutes to mix.

3. Take out the centrifuge tube, and after cooling to room temperature (25°C), add an equal volume of 700 μl of phenol (350 μl)/chloroform (350 μl). Invert up and down to mix well and extract for 5 minutes.

4. Centrifuge at 12,000 rpm for 10 minutes at room temperature, and transfer the supernatant to another new centrifuge tube with a 1ml pipette tip with the head cut off. Add 2 μl RNaseA (10mg/ml).

5. Add 700 μl of chloroform equal to the volume of the supernatant, mix thoroughly by inverting up and down, and extract for 5 minutes.

6. Centrifuge at 12,000 rpm for 10 minutes at room temperature, and transfer the supernatant to another new centrifuge tube.

7. Add an equal volume of isopropanol (700 μl), mix well, and leave at room temperature for 10 minutes to see precipitation. For complete precipitation, it can be placed at -20°C for 1-2 hours.

8. Centrifuge at 12,000 rpm for 10 minutes at room temperature, and the sediment will settle at the bottom of the tube. Discard the supernatant.

9. Add 700 μl of 70% (volume percent) ethanol aqueous solution to wash for 30 minutes.

10. Pour off the ethanol solution in the centrifuge tube, and let the DNA precipitate dry naturally in the tube.

11. Add an appropriate amount of TE (50 μl) to dissolve, and store in a -20°C refrigerator for later use.

2. Real-time fluorescent PCR

Real-time fluorescent PCR reaction system: 12.5 μl AB TaqMan Gene Expression Master Mix, 5 μl DNA template, 5 μl sterilized ultrapure water, 1 μl upstream primer (RB-F) in the kit, 1 μl downstream primer (RB-R) in the kit ) and 0.5 μl of the specific probe in the kit were mixed to obtain a 25 μl reaction system; in the reaction system, at the beginning of the reaction, the concentration of the upstream and downstream primers was 0.2 μmol/L, and the concentration of the probe was 0.1 μmol/L.

Real-time fluorescent PCR reaction parameters: 50°C, 2min; 95°C, 10min; 95°C, 15s, 60°C, 1min, a total of 40 cycles.

After the reaction, judge the result according to the amplification curve.

The results are shown in Figure 1. Only Kefeng No. 6 had fluorescence signal, and the other samples had no fluorescence signal.

Embodiment 3, the sensitivity detection of kit

Take Kefeng No. 6 seed powder and Minghui 86 seed powder as test materials, mix the two kinds of seed powders in different proportions, and obtain a series of samples (the weight percentage of Kefeng No. 6 seed powder in the sample is 5 %, 1%, 0.5%, 0.1% and 0.01%).

The total DNA of each sample was extracted, and 20ng of total DNA was used as a template for real-time fluorescent PCR reaction. The extraction of the total DNA of the sample and the method of real-time fluorescent PCR are the same as in Example 2.

The result is shown in Figure 2. The results show that the method of the present invention can detect at least 0.01% of the transgenic rice line Kefeng 6 (2×10 ^-3 ng).

sequence listing

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Claims (6)

1. Auele Specific Primer is to detecting in preparation whether paddy rice to be measured is the application in rich No. 6 test kit of transgenic paddy rice strain section with probe; Said Auele Specific Primer is right to the primer of forming for Nucleotide shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table, and the nucleotide sequence of said probe is shown in the sequence 3 of sequence table.

2. application as claimed in claim 1 is characterized in that: said specific probe is the TaqMan fluorescent probe, and 5 ' end of said specific probe is marked with the FAM fluorophor, and 3 ' end is marked with the TAMRA quenching group.

3. one kind is detected whether paddy rice to be measured is rich No. 6 test kit of transgenic paddy rice strain section; It comprises the right and specific probe of nucleotide sequence shown in the sequence 3 of sequence table of Auele Specific Primer that Nucleotide is formed shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table.

4. test kit as claimed in claim 3 is characterized in that: said specific probe is the TaqMan fluorescent probe, and 5 ' end of said specific probe is marked with the FAM fluorophor, and 3 ' end is marked with the TAMRA quenching group.

5. whether claim 3 or 4 said test kits are the application in rich No. 6 of the transgenic paddy rice strain section detecting paddy rice to be measured.

6. one kind is detected whether paddy rice to be measured is rich No. 6 method of transgenic paddy rice strain section; Be that genomic dna with paddy rice to be measured is a template; Auele Specific Primer with Nucleotide shown in the sequence 2 of Nucleotide shown in the sequence 1 of sequence table and sequence table is formed is right; Carry out real-time fluorescence PCR with the specific probe of nucleotide sequence shown in the sequence 3 of sequence table and detect, confirm whether paddy rice to be measured is rich No. 6 of transgenic paddy rice strain section; Said specific probe is the TaqMan fluorescent probe, and 5 ' end of said specific probe is marked with the FAM fluorophor, and 3 ' end is marked with the TAMRA quenching group;

In the PCR system of said real-time fluorescence PCR, when reacting initial, the concentration of two primers of said Auele Specific Primer centering is 0.2 μ mol/L, and the concentration of said specific probe is 0.1 μ mol/L; The parameter of said real-time fluorescence PCR is following: 50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s, 60 ℃, 1min, totally 40 circulations.

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