CN103451292A - Identification of specificity of transgenic rice Huahui 1 by applying recombinase polymerase amplification (RPA) technology - Google Patents

Abstract

The invention discloses a primer and probe combination which is suitable for identifying transgenic insect-resistant rice Huahui 1 (TT51-1) by using a recombinase polymerase amplification (RPA) technology. A forward primer sequence of the primer and probe combination is shown by SEQ ID No.1, a reverse primer sequence is shown by SEQ ID No.2, and a probe sequence is shown by SEQ ID No.3. Meanwhile, the invention also discloses a method for identifying the transgenic insect-resistant rice TT51-1. The method comprises the following steps: extracting DNA (Deoxyribonucleic Acid) of a sample to be tested as a template; performing RPA rapid amplification and real-time fluorescence detection by using the primer; if an obvious amplification curve is obtained, proving that the tested sample contains the ingredients of the transgenic insect-resistant rice TT51-1. The invention provides an RPA detection method for the specificity of the transgenic insect-resistant rice TT51-1 for the first time.

Description

Specific Identification of Transgenic Rice Variety Huahui 1 Using RPA Technology

technical field

The invention belongs to the field of biotechnology, and relates to a strain-specific method for identifying the transgenic insect-resistant rice Huahui No. 1 (TT51-1) using the Recombinase Polymerase Amplification (RPA) technology.

Background technique

At present, DNA amplification is the main method of nucleic acid detection. The commonly used PCR detection requires sophisticated instruments and cumbersome test procedures, which is difficult to meet the requirements of on-site detection in non-laboratory environments. In recent years, nucleic acid constant temperature amplification technology has been greatly developed. Compared with traditional PCR, nucleic acid constant temperature amplification technology does not require expensive PCR equipment, and can quickly amplify the target fragment in a short time. It is simple, fast, sensitive, etc. advantage. RPA technology is developed by simulating DNA replication in organisms and based on the principle of recombinase-polymerase-mediated amplification. When a fully complementary sequence is found on the template DNA by using recombinase and primers to form microfilaments, the single-stranded DNA With the help of the binding protein, the template DNA is melted, and the primer and the template DNA begin to pair to form the free 3' hydroxyl end required for replication. Under the action of DNA polymerase, the replication and extension are carried out to form a new DNA complementary chain reaction product. Exponential growth. Unlike conventional PCR reactions, the length of primers required for RPA reactions is usually 30-35nt. In order to avoid the formation of secondary structures within and between primers during primer design, the increase in their length also increases the difficulty of primer design and selection. The design and selection of primer sequences are crucial to the results of RPA. Real-time monitoring of template amplification can be realized by adding a fluorescently labeled probe to the RPA amplification system. The two T bases in the middle of the probe are each labeled with a fluorescent group (FAM and BHQ1). There is an abasic site (dSpacer), which can be recognized by an exonuclease from Escherichia coli, which has 3'-5' exonuclease activity, which can separate the two fluorescent groups, thereby The fluorescent signal is synchronized with the accumulation of the amplified product. Combined with a portable fluorescence amplification detector, the fluorescence curve can be detected within 10-20 minutes. RPA technology greatly shortens the detection time, simplifies the reaction procedure, and combines with DNA rapid extraction technology to make field detection possible, and has a wide range of application prospects.

The methods for detection of transgenic products by PCR technology include screening detection, gene specific detection, construction specific detection and transformant specific detection. Due to the uniqueness of the exogenous gene at the insertion site of the recipient genome, it is highly specific to design RPA primers for detection based on the connection region between the exogenous inserted DNA sequence and the rice genome, and can specifically detect the transformant and its derivatives.

In August 2009, the Ministry of Agriculture issued the safety certificate of transgenic insect-resistant rice TT51-1 in Hubei Province, which aroused widespread public concern about its safety. At present, the commercial planting of TT51-1 has not been approved in my country, but its spread occurs from time to time, and it is urgent to establish a quick and simple detection method for market supervision and routine monitoring.

At present, among the reported detection methods of transgenic plants, the PCR instrument is mainly used for routine detection in the laboratory, and this method cannot further satisfy the rapid detection of genetically modified products. At present, there is no strain-specific identification of transgenic rice Huahui 1 (TT51-1) using RPA technology at home and abroad.

Contents of the invention

Aiming at the gap in the above fields, the present invention provides an accurate, fast and simple RPA detection method for detecting the strain specificity of the transgenic rice Huahui No. 1 (TT51-1).

The technical solution provided by the present invention is: a primer for identifying the transgenic insect-resistant rice Huahui No. 1 (TT51-1) through the isothermal amplification technology of recombinase polymerase, and its forward primer sequence is as SEQ ID No.1 As shown, the reverse primer sequence is shown in SEQ ID No.2, and the probe sequence is shown in SEQ ID No.3.

The present invention also provides a kit for identifying the transgenic insect-resistant rice Huahui No. 1 through the isothermal amplification technology of recombinase polymerase, the kit includes the above-mentioned primers and probes.

The present invention also provides a method for identifying transgenic insect-resistant rice TT51-1 through the isothermal amplification technology of recombinase polymerase: extract the DNA of the sample to be tested as a template, and use the primers described in claim 1 to perform rapid fluorescence detection, if Obvious amplification curves are obtained, which proves that the tested sample is the transgenic insect-resistant rice Huahui 1. The implementation steps are as follows: add 2 mL of primers (10 mmol/L), 0.5 mL of probes (10 mmol/L), and 50 ng of template DNA to the 50 mL amplification system recommended by the RPA amplification kit. RPA amplification detector (or fluorescent quantitative PCR instrument) reacted at 39 degrees Celsius for 15 minutes.

The method of the present invention is to design a large number of RPA-specific primers according to the connection region between the exogenously inserted DNA sequence and the rice genome, and screen out a set of primers and probes that can quickly and effectively detect the components of the transgenic rice Huahui No. 1 (TT51-1) combination. Using this pair of primers for rapid fluorescence detection, using the genomic DNA of the transgenic rice Huahui 1 (TT51-1) as a template, an obvious amplification curve can be obtained, while the non-transgenic rice Minghui 63 and other transgenic rice lines Kefeng 6, etc. There were no amplification curves when the genomic DNA of the three rice materials was amplified as a template. Dilute the template with water to 2000, 400, 200, 100, and 20 copies, and the results all have amplification curves. This method has high sensitivity. For the first time, this invention provides a strain-specific RPA for the transgenic insect-resistant rice Huahui No. 1 (TT51-1) Detection method. The method enables improved capabilities for accurate and rapid detection of biotechnology products.

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Description of drawings

Figure 1 is a specific detection map of TT51-1, in which, 1: transgenic rice TT51-1; 2: transgenic rice Kefeng No. 6; 3: transgenic rice Kemodao 1; 4: non-transgenic rice Minghui 63; 5: water

Figure 2 is the sensitivity test diagram, the template copy numbers from 1 to 6 are 2000; 400; 200; 100; 20; 0

Detailed ways

The present invention will be further clarified through the detailed description of specific embodiments below, but it is not intended to limit the present invention, but only for illustration.

The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions, such as the conditions described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 2001) by Sambrook et al., or in accordance with the conditions of the instrument Or the conditions recommended by the reagent manufacturer.

First, design primers: design primers and probes according to the specific sequence of TT51-1 transformant (GenBank No. EU880444). RPA requires a primer length of 30-35nt, which will easily lead to a large number of primer dimers under constant temperature conditions, which will affect the experimental effect. RPA experiments need to design multiple pairs of primers from both ends of the target sequence for optimization, screening, and individual bases The replacement or increase or decrease will have an important impact on the experimental results. A large number of primers were designed in this experiment, and a pair of primers with high sensitivity and specificity were screened out for RPA fluorescence detection. The primers and probe sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 (Table 1).

Note: FAM: luminescent group; dSpacer: abasic site; BHQ1: quencher group; phosphate: phosphate group

1. Experimental Materials

(1) Plant material

The transgenic rice TT51-1, the genetically modified rice Kefeng 6, the genetically modified rice Kechidao 1, and the non-transgenic rice Minghui 63.

(2) Enzymes and reagents

Molecular biology reagents, TwistAmp DNA Amplification Exo Kits were purchased from TwistDX Company, and other biochemical reagents were imported or domestic analytically pure. Primers were synthesized by Beijing Sangon Biotechnology Co., Ltd.

(3) Experimental equipment

DNA processing instrument: low temperature mixing ball mill MM400 (Retsch)

Fluorescence detector: RPA amplification detector (Twista) or fluorescent quantitative PCR instrument.

Other instruments include: constant temperature water bath, electronic balance, centrifuge, vortex instrument, pure water instrument, constant temperature incubator, etc.

2. Experimental Methods and Procedures

(1) Extraction of rice genomic DNA

Rice single-grain planting, the young leaves were taken as DNA extraction materials, and the total DNA of rice was extracted according to the operation manual of the TianGen Plant Genomic DNA Kit (Cat#DP-305) kit.

(2) DNA concentration and purity determination

The purity and concentration of DNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific), and the DNA concentration was adjusted to 50 ng/mL with deionized double-distilled water.

(3) Primer amplification

In this example, the PRA method was used to amplify the transformant-specific sequence between the exogenous inserted DNA and the plant genomic DNA to identify the transgenic rice TT51-1, and the template concentration was 50 ng/mL.

The RPA amplification system is: total system 50mL, add 29.5mL of rehydration buffer, 2.5mL of magnesium acetate solution (280mmol/L), 2mL of each primer (10mmol/ L), probe 0.5mL (10mmol/L), template DNA 50ng, make up the rest with water;

Primer amplification program: RPA amplification detector reacted at 39 degrees Celsius for 15 minutes.

3. Experimental results

The forward primer and reverse primer designed according to the specific sequence of the transformant were used for RPA fluorescence detection on the genomic DNA of Minghui 63, Kefeng 6, Kechidao 1 and TT51-1 rice, which can quickly and accurately identify the transgenic rice TT51 -1, in which the transgenic rice TT51-1 had an obvious fluorescence curve, while the non-transgenic rice Minghui 63 and the transgenic rice Kefeng 6 had no amplification curve (Figure 1). Dilute the template with water to 2000, 400, 200, 100, and 20 copies, and the results all have amplification curves (Figure 2). It shows that the identification of transgenic rice TT51-1 by using the primers and method of the present invention has high sensitivity and accuracy, and is easy to operate.

<110>Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> Application of RPA technology to specific identification of transgenic rice line Huahui No. 1

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Claims (4)

1. A combination of primers and probes for identifying the transgenic insect-resistant rice Huahui No. 1 by recombinase polymerase isothermal amplification technology, characterized in that: its forward primer sequence is as shown in SEQ ID No.1, The reverse primer sequence is shown in SEQ ID No.2, and the probe sequence is shown in SEQ ID No.3. 2. A kit for identifying the transgenic insect-resistant rice variety Huahui No. 1, characterized in that the kit comprises the combination of primers and probes according to claim 1. 3. A method for identifying transgenic insect-resistant rice Huahui No. 1 by recombinase polymerase isothermal amplification technology, characterized in that: the DNA of the sample to be tested is extracted as a template, and the primers and probes according to claim 1 are used Combining rapid amplification and real-time fluorescence detection, if an obvious amplification curve is obtained, it proves that the tested sample contains components of the transgenic insect-resistant rice Huahui No. 1. 4. The method of claim 3, wherein: Add 2mL of 10mmol/L primers, 0.5mL of 10mmol/L probes, and 50ng of template DNA to the 50mL amplification system recommended by the RPA amplification kit; The amplification program is: RPA amplification detector or fluorescent quantitative PCR instrument reacts at 39 degrees Celsius for 15 minutes.

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