CN105483236A - LAMP (Loop-mediated isothermal amplification) detection primer group and kit and detection method for gene cry1C of transgenic plants - Google Patents

Abstract

The invention discloses a LAMP detection primer set, kit and detection method for <i>cry1C</i> gene in transgenic plants. The primer set consists of 6 specific primers, the nucleotide sequence of which is SEQ? ID? NO: 1~6 shown. The detection kit includes detection primer solution, DNA polymerase with strand displacement activity, 10× reaction buffer, dNTPs solution, and chromogenic reagent. The detection method is to use specific primers and DNA polymerase with strand displacement activity to amplify the sample DNA template at 63~65°C, and add a chromogenic agent to observe the color change to determine whether the sample contains <i>cry1C </i> Genetic component. The invention does not require special instruments, has the characteristics of rapidity, high efficiency, simple operation, strong specificity, etc., and is suitable for on-site detection.

Description

LAMP detection primer set, kit and detection method for cry1C gene in transgenic plants

technical field

The invention belongs to the technical field of molecular biology, and relates to a detection method for transgenic plants and their products, in particular to a primer set, a kit and a detection method for rapidly detecting cry1C gene in transgenic plants using loop-mediated isothermal amplification technology (LAMP) .

Background technique

In 2014, the global planting area of genetically modified crops reached 181.5 million hectares, an increase of more than 100 times compared with the initial commercialization in 1996. Currently commercially grown genetically modified crops mainly include soybean, corn, cotton, and rapeseed, and the main traits are resistance to insects and herbicides. The insect resistance gene derived from Bacillus thuringiensis ( Bt ) is currently the most widely used insect resistance gene in transgenic plants, including cry1Ab , cry1F, cry1Ie , cry1C , cry34Ab, cry35Ab, vip3A, etc. Among them, the cry1C gene is independently produced by our country. A high-efficiency insect-resistant gene developed has been widely used in the research of new varieties of transgenic corn and soybeans. Research on rapid and accurate detection methods for the cry1C gene is the technical support and guarantee for the smooth implementation of laws and regulations related to the safety management of genetically modified organisms.

Genetically modified component detection technology is mainly divided into two categories: one is exogenous DNA as the detection object, such as PCR, gene chip, etc., and the other is the protein expressed by exogenous gene as the detection object, such as ELISA, immune test strips, etc. Among the above-mentioned methods, PCR technology is currently the most widely used, but PCR-based transgenic detection methods require professional equipment such as PCR machines and gel imaging systems, and the time for amplification and product detection is long (about 3-4 hours), which is difficult to achieve. The purpose of rapid on-site detection, therefore, a more convenient, accurate, and on-site transgenic detection technology is needed in practical work.

LAMP technology is a new gene amplification technology developed by Japan's Eiken Chemical Co., Ltd. in 2000. The basic characteristics of this technology are: ① constant temperature amplification: the entire amplification reaction is carried out under constant temperature conditions (60~65°C) , no special equipment is required; ②fast and efficient: the entire amplification and product detection can be completed within 1 hour; ③high specificity: 4 detection primers are designed for 6 regions of the target sequence, and the amplification specificity is high; ④high sensitivity : The detection limit can be as low as 10 copies or lower; ⑤Easy identification: the precipitation change in the reaction tube can be observed with the naked eye or a turbidimeter, or the color change can be observed with the naked eye after adding a dye to the reaction tube. The product can be easily detected.

The LAMP method has the characteristics of fast, efficient, easy to operate, strong specificity, and high sensitivity. It does not require special instruments and is suitable for on-site rapid detection. It has broad application prospects in the field of transgenic plant detection. At present, there is no detection method and kit for detecting cry1C gene in transgenic plants by using the LAMP method.

Contents of the invention

One object of the present invention is to disclose a primer set for LAMP detection of cry1C gene in transgenic plants.

Another object of the present invention is to disclose a LAMP detection kit for cry1C gene in transgenic plants.

Another object of the present invention is to disclose a LAMP detection method for detecting cry1C gene in transgenic plants based on the above primer set and kit.

The technical scheme adopted in the present invention is as follows:

According to the nucleotide sequence of the cry1C gene, a primer set for LAMP detection of the cry1C gene was designed, the nucleotide sequence of which was shown in SEQ ID NO: 1-6; the technical parameters of the LAMP detection method were determined, and the specificity of the detection method was verified.

The LAMP detection primer set of cry1C gene in transgenic plants includes outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB, and its nucleotide sequences are as follows:

Outer primer F3: CTTTAGGATCTCTGGATTTGAAG (SEQ ID NO: 1);

Outer primer B3: TGTAAGTGTTAGCACAGTGA (SEQ ID NO: 2);

Internal primer FIP: TCCGAAGATCACAGAATCTCTAAGGTTCCACTTCCTCTCTGTTTACG (SEQ ID NO: 3);

Internal primer BIP: GATGGGGATTGACAACCATCAAACTCATCGATGTGCCTGAT (SEQ ID NO: 4);

Loop primer LF: AAGATGGAGATTAGCAGCTTGAG (SEQ ID NO: 5);

Loop primer LB: CGTGAACGAGAACTACAACAGAC (SEQ ID NO: 6).

LAMP detection kit for cry1C gene in transgenic plants, including the following components:

(1) Detection primer solution: the detection primer solution prepared by the above 6 primers, the concentrations of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF and loop primer LB are 4~6 μmol/L in sequence , 4~6μmol/L, 32~48μmol/L, 32~48μmol/L, 4~6μmol/L and 4~6μmol/L;

(2) BstDNA polymerase with strand displacement activity: the concentration is 7~9U/μL;

(3) 10× reaction buffer: 200mmol/LTris-HCl, pH8.8, 100mmol/LKCl, 100mmol/L(NH ₄ ) ₂ SO ₄ , 40~100mmol/LMgSO ₄ , 6~14mol/L betaine;

(4) dNTPs solution, which is formed by mixing equal volumes of four deoxyribonucleotide solutions of dATP, dCTP, dGTP, and dTTP with a concentration of 10 mmol/L;

(5) Chromogen: 1000×SYBRGREENI fluorescent dye.

Preferably, the detection primer solution contains 5 μmol/L outer primer F3, 5 μmol/L outer primer B3, 40 μmol/L inner primer FIP, 40 μmol/L inner primer BIP, 5 μmol/L loop primer LF, 5 μmol/L loop primer Primer LB.

Preferably, the concentration of the BstDNA polymerase is 8U/μL.

Preferably, the concentrations of MgSO ₄ and betaine in the 10× reaction buffer are 80mmol/L and 8mol/L respectively.

The method for detecting the cry1C gene using the kit described above comprises the following steps:

(1) Extract the genomic DNA of the sample to be tested;

(2) Prepare the LAMP detection reaction system for the sample to be tested: add 2~5 μL of template DNA, 1 μL of detection primer solution, 1 μL of BstDNA polymerase, 2.5 μL of 10× reaction buffer, and 2~5 μL of dNTPs solution in a 200 μL PCR reaction tube, and use a sterile Make up to a total volume of 25 μL with deionized water or ultrapure water;

(3) Add 1 μL of chromogen to the cap of the reaction tube and cover it on the cap of the reaction tube;

(4) Run the LAMP amplification reaction: place the reaction tube in a constant temperature water bath, incubate at 65°C for 60 minutes, and incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Invert the reaction tube to fully mix the chromogen on the tube cap with the reaction solution. If the reaction tube turns green, it is positive, and if it turns orange, it is negative.

It is proved by experiments that the LAMP detection primer set, kit and detection method of cry1C gene provided by the present invention have the advantages of rapidity, high efficiency, simple operation, strong specificity and the like.

Description of drawings

Figure 1 is the results of specific detection in Example 2, 1 to 8 are the cry1Ie gene transgenic corn IE09S034, cry1C gene transgenic rice T1c-19, cry1F gene transgenic corn TC1507, cry34Ab and cry35Ab gene transgenic corn 59122, vip3A transgenic Gene MIR162, dmo transgenic soybean MON87708, aad1 transgenic maize DAS40278-9 and non-transgenic maize control.

detailed description

The present invention will be further described below in conjunction with embodiment.

Embodiment 1 kit and detection method thereof.

Prepare the LAMP detection kit of cry1C gene according to the following formula, and the specification of each kit is 100 reactions:

(1) Detection primer solution: Synthesize outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer LF, and loop primer LB, and make primer dry powder into mother solutions with a concentration of 100 μmol/L with ultrapure water, Then take 5 μL of outer primer F3, 5 μL of outer primer B3, 40 μL of inner primer FIP, 40 μL of inner primer BIP, 5 μL of loop primer LF, and 5 μL of loop primer LB, put them into a new 1.5mL centrifuge tube, mix well, and prepare 100 μL of cry1C gene LAMP detection primer solution, where the primer sequences are:

Outer primer F3: CTTTAGGATCTCTGGATTTGAAG (SEQ ID NO: 1);

Outer primer B3: TGTAAGTGTTAGCACAGTGA (SEQ ID NO: 2);

Internal primer FIP: TCCGAAGATCACAGAATCTCTAAGGTTCCACTTCCTCTCTGTTTACG (SEQ ID NO: 3);

Internal primer BIP: GATGGGGATTGACAACCATCAAACTCATCGATGTGCCTGAT (SEQ ID NO: 4);

Loop primer LF: AAGATGGAGATTAGCAGCTTGAG (SEQ ID NO: 5);

Loop primer LB: CGTGAACGAGAACTACAACAGAC (SEQ ID NO: 6);

(2) BstDNA polymerase: the concentration is 8U/μL, aliquot 100μL into a new 1.5mL centrifuge tube;

(3) 10× reaction buffer: containing 200mmol/LTris-HCl, pH8.8, 100mmol/LKCl, 100mmol/L(NH ₄ ) ₂ SO ₄ , 80mmol/LMgSO ₄ and 8mol/L betaine, dispensed into 250μL into a new 1.5mL centrifuge tube;

(4) dNTPs solution, which is made by mixing equal volumes of four deoxyribonucleotide solutions of dATP, dCTP, dGTP, and dTTP with a concentration of 10mmol/L, and put 500μL into a new 1.5mL centrifuge tube;

(5) Chromogen: 1000×SYBRGREENI fluorescent dye, aliquot 200μL into a new brown 1.5mL centrifuge tube.

Use the above kit to detect the sample to be tested in the following way:

(1) Extract the genomic DNA of the sample to be tested: use the plant DNA extraction kit method produced by Beijing Tiangen Company to extract the genomic DNA of the sample to be tested and dilute to 25ng/μL;

(2) Prepare the LAMP detection reaction system for the sample to be tested: use a 25 μL reaction system, add 15.5 μL of ultrapure water, 2 μL of template DNA, 1 μL of detection primer solution, 1 μL of BstDNA polymerase, and 2.5 μL of 10× reaction buffer in a 200 μL reaction tube. μL, dNTPs solution 3 μL;

(3) Add 1 μL of chromogen to the cap of the reaction tube and cover it on the cap of the reaction tube;

(4) Run the LAMP amplification reaction: place the reaction tube in a constant temperature water bath, incubate at 65°C for 60 minutes, and incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Invert the reaction tube to fully mix the chromogen on the tube cap with the reaction solution. If the reaction tube turns green, it is positive, and if it turns orange, it is negative.

Embodiment 2 The specificity experiment of kit and detection method.

Prepare the kit according to the kit preparation method described in Example 1, and carry out the specificity test for its detection method:

(1) Extraction of cry1Ie-transgenic maize IE09S034, cry1C-transgenic rice T1c-19, cry1F-transgenic maize TC1507, cry34Ab and cry35Ab-transgenic maize 59122, vip3A-transgenic MIR162, dmo-transgenic soybean MON87708, aad1-transgenic maize DAS40278-9 Genomic DNA of 8 kinds of test materials such as non-transgenic corn and non-transgenic corn, the DNA concentration was measured with ND1000 nucleic acid micrometer, and diluted to 25ng/μL with ultrapure water;

(2) In the 200 μL eight-tube tube, according to the steps described in Example 1, add 15.5 μL of ultrapure water, 1 μL of detection primer solution, 1 μL of BstDNA polymerase, and 2.5 μL of 10× reaction buffer to each tube in sequence , 3 μL of dNTPs solution, and then add 2 μL of IE09S034, T1c-19, TC1507, 59122, MIR162, MON87708, DAS40278-9, and DNA controls of non-transgenic corn samples to tubes 1-8 respectively;

(3) Add 1 μL of chromogenic reagent to each cap of the eight-tube tube cap, and cover it on the eight-tube tube;

(4) Run the LAMP amplification reaction: place the eight tubes in a constant temperature water bath, incubate at 65°C for 60 minutes, and incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Invert the reaction tube to fully mix the chromogen on the cap of the eight-tube tube with the reaction solution. If the reaction tube appears green, it is positive, and if it appears orange, it is negative.

In this example, only the transgenic rice T1c-19 sample tube containing the cry1C gene appeared green, indicating that the kit and detection method of the present invention have good specificity for the cry1C gene.

It should be noted that the above-mentioned embodiments are specific implementation examples of the present invention, but the implementation mode of the present invention is not limited by the above-mentioned embodiments, and those of ordinary skill in the art can directly derive or associate from the content disclosed in the present invention All deformations should be considered within the protection scope of the present invention.

Claims (6)

1. in transgenic plant cry1Cthe LAMP detection primer group of gene, comprises outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB, and its nucleotide sequence is as follows respectively:

Outer primer F3:CTTTAGGATCTCTGGATTTGAAG(SEQIDNO:1);

Outer primer B3:TGTAAGTGTTAGCACAGTGA(SEQIDNO:2);

Inner primer FIP:TCCGAAGATCACAGAATCTCTAAGGTTCCACTTCTCTCTGTTTACG(SEQID NO:3);

Inner primer BIP:GATGGGGATTGACAACCATCAAACTCATCGATGTGCCTGAT(SEQIDNO:4);

Ring primer LF:AAGATGGAGATTAGCAGCTTGAG(SEQIDNO:5);

Ring primer LB:CGTGAACGAGAACTACAACAGAC(SEQIDNO:6).

2. in transgenic plant cry1Cthe LAMP detection kit of gene, comprises following component:

(1) detect primer solution: the detection primer solution prepared by 6 primers according to claim 1, the concentration of outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, ring primer LF and ring primer LB is followed successively by 4 ~ 6 μm of ol/L, 4 ~ 6 μm of ol/L, 32 ~ 48 μm of ol/L, 32 ~ 48 μm of ol/L, 4 ~ 6 μm of ol/L and 4 ~ 6 μm ol/L;

(2) there is the BstDNA polysaccharase of strand-displacement activity: concentration is 7 ~ 9U/ μ L;

(3) 10 × reaction buffers: 200mmol/LTris-HCl, pH8.8,100mmol/LKCl, 100mmol/L (NH ₄) ₂sO ₄, 40 ~ 100mmol/LMgSO ₄, 6 ~ 14mol/L trimethyl-glycine;

(4) dNTPs solution, dATP, dCTP, dGTP, dTTP tetra-kinds of deoxyribonucleotide solution equal-volumes being respectively 10mmol/L by concentration mix;

(5) developer: 1000 × SYBRGREENI fluorescence dye.

3. according to claim 2 cry1Cthe LAMP detection kit of gene, is characterized in that: containing 5 μm of ol/L outer primer F3,5 μm of ol/L outer primer B3,40 μm of ol/L inner primer FIP, 40 μm of ol/L inner primer BIP, 5 μm of ol/L ring primer LF, 5 μm of ol/L ring primer LB in described detection primer solution.

4. according to claim 2 cry1Cthe LAMP detection kit of gene, is characterized in that: the concentration of described BstDNA polysaccharase is 8U/ μ L.

5. according to claim 2 cry1Cthe LAMP detection kit of gene, is characterized in that: MgSO in 10 described × reaction buffer ₄, beet paper mill wastewater is respectively 80mmol/L, 8mol/L.

6. utilize the test kit described in any one of claim 2 ~ 5 to detect cry1Cthe method of gene, comprises the following steps:

(1) genomic dna of testing sample is extracted;

(2) the LAMP detection reaction system of testing sample is prepared: in 200 μ L reaction tubess, adding template DNA 2 ~ 5 μ L, detect primer solution 1 μ L, BstDNA polysaccharase 1 μ L, 10 × reaction buffer 2.5 μ L, dNTPs solution 2 ~ 5 μ L, is 25 μ L with sterilizing deionized water or ultrapure water polishing to cumulative volume;

(3) cover the developer adding 1 μ L at reaction tubes, cover on reaction tubes;

(4) LAMP amplified reaction is run: be placed on by reaction tubes in thermostat water bath, hatch 60min for 65 DEG C, hatch 5min termination reaction for 80 DEG C;

(5) qualification of LAMP amplification: put upside down reaction tubes, the developer that pipe is covered fully mixes with reaction soln, if reaction tubes shows green, is positive, if manifest orange, is negative.