CN103924000B - LAMP detecting primer group, kit and detecting method for cry3A gene in transgenic plant - Google Patents

Abstract

The invention discloses a LAMP detection primer set, a kit and a detection method for cry3A gene in transgenic plants. The primer set is composed of 5 specific primers, the nucleotide sequences of which are shown in SEQ ID NO: 1-5. The detection kit includes a detection primer solution, a loop primer solution, a DNA polymerase with strand displacement activity, a 10× reaction buffer, a dNTPs solution, a positive DNA control and a negative DNA control, and the kit may also contain a 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 to observe the color change by adding a chromogenic agent or directly observe the turbidity change of the precipitate in the reaction tube, etc. The method is to determine whether the sample contains the cry3A gene component. The invention does not require special instruments, has the characteristics of rapidity and high efficiency, simple and convenient operation, strong specificity, high sensitivity, etc., and is suitable for on-site detection.

Description

LAMP detection primer set, kit and detection method for cry3A 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 products thereof, in particular to a primer set, a kit and a detection method for rapidly detecting cry3A gene in transgenic plants using loop-mediated isothermal amplification technology (LAMP) .

Background technique

In 2013, the global planting area of genetically modified crops reached 175.2 million hectares, an increase of more than 100 times compared with the initial commercialization in 1996. At present, commercially grown transgenic crops mainly include soybean, corn, cotton, and rapeseed, and the main traits are resistance to insects and herbicides. The target genes used in insect-resistant transgenic plants include cry1Ab , cry1Ac , cry1A.105 , cry1F, cry2Ab , cry3A , cry3Bb, vip3A, cry9C, etc. Among them, the cry3A gene exists in the transgenic maize MIR604, which has been approved in the United States, the European Union, and Australia , Russia, South Korea, South Africa and other nearly 20 countries and regions commercially planted or used as food and feed, China also approved the import of genetically modified corn MIR604 in 2008 to be used as processing raw materials.

In order to strengthen the safety management of GMOs and their products, more than 50 countries and regions around the world have formulated GMO labeling systems. In 2001, my country promulgated and implemented the "Regulations on the Safety Management of Agricultural Genetically Modified Organisms" and a series of supporting management measures such as safety evaluation, labeling management, import safety, and processing approval. Mandatory labeling management system for 17 kinds of products in 5 categories including rapeseed, cotton and tomato.

GMO detection technology is an important technical support for the safety management of GMOs. At present, GMO 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, the PCR method is the most widely used method, but the PCR-based transgenic detection method requires professional equipment such as a PCR machine and a gel imaging system, and the time for amplification and product detection is long (about 3-4 h), which is difficult to detect. To achieve 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 less; ⑤Easy identification: There are many identification methods for amplified products, such as visual observation or using a turbidimeter to observe the turbidity change of the precipitate in the reaction tube, adding dyes to observe the color change, etc. .

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 cry3A gene in transgenic plants by using the LAMP method.

Contents of the invention

One object of the present invention is to disclose a LAMP detection primer set for cry3A gene in transgenic plants.

Another object of the present invention is to disclose a LAMP detection kit for cry3A gene in transgenic plants.

Another object of the present invention is to disclose a LAMP detection method for detecting cry3A 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 cry3A gene, design the LAMP detection primer set of cry3A gene, its nucleotide sequence is as SEQ ID NO: 1~5; Determine the technical parameters of the LAMP detection method, and the specificity and sensitivity of the detection method verify.

The primer set for LAMP detection of cry3A gene in transgenic plants includes outer primer 1, outer primer 2, inner primer 1, inner primer 2, and loop primer. The nucleotide sequences are as follows:

Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1);

Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2);

Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3);

Internal primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4);

Loop primer: GGGCTGGAAACGCGTGT (SEQ ID NO: 5).

LAMP detection kit for cry3A gene in transgenic plants, including the following components:

(1) Detection primer solution: detection primer solution prepared by 2 outer primers and 2 inner primers, the concentrations of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 are 4~6 μmol/L, 4 ~6 μmol/L, 32~48 μmol/L and 32~48 μmol/L;

(2) Loop primer solution: the concentration of loop primer is 16~24 μmol/L;

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

(4) 10× reaction buffer: 200 mmol/L Tris-HCl (pH 8.8), 100 mmol/L KCl, 100 mmol/L (NH ₄ ) ₂ SO ₄ , 40~100 mmol/L MgSO ₄ , 6~ 14 mol/L betaine;

(5) 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;

(6) Positive DNA control samples, genomic DNA of cry3A transgenic plants, or Escherichia coli plasmid DNA containing cry3A gene sequence;

(7) Negative DNA control sample: DNA without cry3A gene sequence.

Preferably, the detection primer solution contains 5 μmol/L outer primer 1, 5 μmol/L outer primer 2, 40 μmol/L inner primer 1, and 40 μmol/L inner primer 2.

Preferably, the loop primer solution contains 20 μmol/L loop primer.

Preferably, the DNA polymerase with strand displacement activity is Bst DNA polymerase with a concentration of 8 U/μL.

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

The LAMP detection kit for the cry3A gene of the present invention may also contain a chromogen, which is a SYBR GREEN I fluorescent dye.

The method for detecting the cry3A gene using the kit described above comprises the following steps:

(1) Extract the genomic DNA of the sample to be tested: extract the genomic DNA of the sample to be tested according to the conventional CTAB method or the plant genomic DNA extraction kit;

(2) Prepare the LAMP detection reaction system for the sample to be tested: add 2~5 μL of template DNA, 1.5 μL of detection primer solution, 1 μL of loop primer solution, 1 μL of Bst DNA polymerase, and 10× reaction buffer into a 200 μL PCR reaction tube solution 2.5 μL, dNTPs solution 3 μL, make up to 25 μL with sterilized deionized water;

(3) Prepare the LAMP detection reaction system of the control sample: When preparing the positive control and negative control reaction systems, except that the template DNA is replaced with the positive DNA control sample and the negative DNA control sample, the other components are consistent with step (2);

(4) Run the LAMP amplification reaction: incubate at 63-65°C for 30-60 minutes, incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Judging the amplification results by visual observation or using a turbidimeter to observe the turbidity change of the precipitate in the reaction tube, or take 2~5 μL of amplification products for agarose gel electrophoresis detection amplification result.

If the kit contains a chromogenic reagent, add 1-2 μL of chromogenic reagent to the LAMP amplification product obtained after step (4), and judge the amplification result by visually observing the color development result.

Experiments have proved that the LAMP detection primer set, kit and detection method for the cry3A gene provided by the present invention have the advantages of rapidity, high efficiency, simple operation, strong specificity, and high sensitivity.

Description of drawings

Fig. 1 is the LAMP detection result figure of embodiment 1, and 1 is a positive control sample, 2 is a negative control sample, and 3 and 4 are samples to be tested;

Fig. 2 is the LAMP detection result figure of embodiment 2, and 1 is a positive control sample, 2 is a negative control sample, and 3 and 4 are samples to be tested;

Figure 3 is the results of specific detection in Example 3, 1-15 are transgenic corn MON89034, MON810, Bt11, Bt176, transgenic cotton MON531, MON15985, transgenic rice KF-6, TT51-1, transgenic corn MON863, MON88017, MIR604, TC1507, 59122, non-transgenic corn negative control, blank control;

Fig. 4 and Fig. 5 are the result graphs of carrying out sensitivity detection in embodiment 4, and 1～7 are respectively 100%, 10%, 1%, 0.1%, 0.05%, 0..02% of mass fraction of transgenic corn MIR604, 0.01% of the test samples, 8 is the non-transgenic corn sample, and M is the DNA molecular weight marker.

Detailed ways

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

Embodiment 1 Kit and detection method thereof without chromogenic agent:

Prepare the LAMP detection kit of cry3A gene according to the following formula:

(1) Detection primer solution: Synthesize outer primer 1, outer primer 2, inner primer 1, and inner primer 2, prepare dry powder of primers with sterilized deionized water to prepare mother solutions with a concentration of 100 μmol/L, and then take 7.5 μL outer primer Primer 1, 7.5 μL outer primer 2, 60 μL inner primer 1, 60 μL inner primer 2, 15 μL sterilized deionized water, and mix thoroughly to prepare 150 μL cry3A gene LAMP detection primer solution, in which the primer sequences are :

Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1);

Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2);

Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3);

Internal primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4);

(2) Loop primer solution: To synthesize loop primers, prepare dry powder of primers with sterilized deionized water to make a mother solution with a concentration of 100 μmol/L, then take 20 μL of loop primers and 80 μL of sterilized deionized water, and mix well to prepare Make 100 μL of cry3A gene LAMP loop primer solution, where the primer sequence is:

Loop primer: GGGCTGGAAACGCGTGT (SEQ ID NO: 5);

(3) Bst DNA polymerase, the concentration is 8 U/μL;

(4) 10× reaction buffer: 200 mmol/L Tris-HCl (pH 8.8), 100 mmol/L KCl, 100 mmol/L (NH ₄ ) ₂ SO ₄ , 80 mmol/L MgSO ₄ , 8 mol/L Betaine;

(5) 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;

(6) Positive DNA control samples, genomic DNA of cry3A transgenic plants, or Escherichia coli plasmid DNA containing cry3A gene sequence;

(7) Negative DNA control sample: DNA without cry3A gene sequence.

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;

(2) Prepare the LAMP detection reaction system for the sample to be tested: add 2 μL of template DNA, 1.5 μL of detection primer solution, 1 μL of loop primer solution, 1 μL of Bst DNA polymerase, and 2.5 μL of 10× reaction buffer into a 200 μL PCR reaction tube. μL, dNTPs solution 3 μL, make up to 25 μL with sterilized deionized water;

(3) Prepare the LAMP detection reaction system of the control sample: When preparing the positive control and negative control reaction systems, except that the template DNA is replaced with the positive DNA control sample and the negative DNA control sample, the other components are consistent with step (2);

(4) Place the reaction tube in the turbidimeter and run the LAMP amplification reaction: incubate at 63°C for 40 minutes, incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Use a real-time turbidimeter (Eiken, Japan) to observe the turbidity changes of the precipitate in the reaction tube (reflected as a real-time amplification curve) to judge the amplification results.

In this example, the positive control had precipitation (a typical amplification curve appeared), and the negative control had no precipitation (no typical amplification curve); the reaction tube of the test sample 1 had precipitation, indicating that it contained the cry3A gene component; the test sample No precipitation appeared in the reaction tube of 2, indicating that the cry3A gene component was not contained.

Embodiment 2 Containing the kit of chromogenic agent and its detection method

Prepare the LAMP detection kit of cry3A gene according to the following formula:

(1) Detection primer solution: Synthesize outer primer 1, outer primer 2, inner primer 1, and inner primer 2, make primer dry powder into mother solutions with a concentration of 100 μmol/L with sterilized deionized water, and then take 7.5 μL outer primer Primer 1, 7.5 μL outer primer 2, 60 μL inner primer 1, 60 μL inner primer 2, 15 μL sterilized deionized water, and mix well to prepare 150 μL cry3A gene LAMP detection primer solution, in which the primer sequences are :

Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1);

Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2);

Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3);

Internal primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4);

(2) Loop primer solution: To synthesize loop primers, prepare dry powder of primers with sterilized deionized water to make a mother solution with a concentration of 100 μmol/L, then take 20 μL of loop primers and 80 μL of sterilized deionized water, and mix well to prepare Make 100 μL of cry3A gene LAMP loop primer solution, where the primer sequence is:

Loop primer: GGGCTGGAAACGCGTGT (SEQ ID NO: 5);

(3) Bst DNA polymerase, the concentration is 8 U/μL;

(4) 10× reaction buffer: 200 mmol/L Tris-HCl (pH 8.8), 100 mmol/L KCl, 100 mmol/L (NH ₄ ) ₂ SO ₄ , 80 mmol/L MgSO ₄ , 8 mol/L Betaine;

(5) 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;

(6) Positive DNA control samples, genomic DNA of cry3A transgenic plants, or Escherichia coli plasmid DNA containing cry3A gene sequence;

(7) Negative DNA control sample: DNA without cry3A gene sequence;

(8) Chromogen: 1000×SYBR GREEN I fluorescent dye. `` ``

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;

(2) Prepare the LAMP detection reaction system for the sample to be tested: add 2 μL of template DNA, 1.5 μL of detection primer solution, 1 μL of loop primer solution, 1 μL of Bst DNA polymerase, and 2.5 μL of 10× reaction buffer into a 200 μL PCR reaction tube. μL, dNTPs solution 3 μL, make up to 25 μL with sterilized deionized water;

(3) Prepare the LAMP detection reaction system of the control sample: When preparing the positive control and negative control reaction systems, except that the template DNA is replaced with the positive DNA control sample and the negative DNA control sample, the other components are consistent with step (2);

(4) Place the reaction tube in a constant temperature water bath and run the LAMP amplification reaction: incubate at 63°C for 40 minutes, incubate at 80°C for 5 minutes to terminate the reaction;

(5) Identification of LAMP amplification results: Add 2 μL of chromogenic reagent to the LAMP amplification product and mix well. If the reaction tube appears green, it is positive, and if it appears orange, it is negative.

In this example, the positive control is green, and the negative control is orange; the reaction tube of test sample 1 is green, indicating that it contains the cry3A gene component; the reaction tube of test sample 2 is orange, indicating that it does not contain the cry3A gene component.

Embodiment 3 The specificity experiment of kit and detection method

The specificity of the kit and its detection method described in Example 2 was tested, and the test samples included: transgenic corn MIR604 ( cry3A ), MON89034 ( cry1A.105 , cry2Ab ), MON810 ( cry1Ab ), Bt11 ( cry1Ab ), Bt176 ( cry1Ab ), MON863 ( cry3Bb ), MON88017 ( cry3Bb ), TC1507 ( cry1F ), 59122 ( cry34Ab , cry35Ab ), transgenic cotton MON531 ( cry1Ac ), MON15985 ( cry1Ac , cry2Ab ), transgenic rice KF-6 ( cry51Ab ), -1 ( cry1Ab/Ac ) and other 13 insect-resistant transgenic crops, as well as non-transgenic corn.

In this example, the reaction tube of the transgenic maize MIR604 sample containing only the cry3A gene appears green, indicating that the kit and detection method of the present invention have good specificity for the cry3A gene.

Embodiment 4 Sensitivity experiment of kit and detection method

Experiments were carried out on the sensitivity of the kit described in Example 2 and its detection method. The test sample was prepared by mixing transgenic corn MIR604 (containing cry3A gene) and non-transgenic corn in a certain mass ratio. MIR604 corn in 8 test samples The mass fractions were 100%, 10%, 1%, 0.1%, 0.05%, 0.02%, 0.01%, and 0, respectively.

In the present embodiment, the MIR604 corn mass fraction is respectively 100%, 10%, 1%, 0.1%, 0.05%, 0.02%, 0.01% of the test sample reaction tubes all appear green, while the negative control appears orange, indicating that the present invention The detection sensitivity of the kit and the detection method thereof can reach 0.01%.

Using the outer primer 1 and outer primer 2 described in the present invention, perform qualitative PCR detection on the above test samples.

The PCR detection reaction system is: 1×PCR buffer (Dalian Bao Biological Company), 0.2 mmol/L dNTPs (Dalian Bao Biological Company), 0.2 μmol/L outer primer 1, 0.2 μmol/L outer primer 2, 1 U Taq DNA Polymerase (Dalian Bao Biological Company), 2 μL template DNA, filled to 25 μL with sterilized deionized water. The PCR reaction program was: pre-denaturation at 94°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 7 min. After PCR, 5 μL of the amplified product was subjected to 2% agarose gel electrophoresis for detection.

In this example, typical electrophoresis bands appeared in the PCR amplification products of the test samples whose mass fractions of MIR604 corn were 100%, 10%, 1%, 0.1%, and 0.05%, while 0.02%, 0.01%, and negative controls had no Typical electrophoresis bands, indicating that the detection sensitivity of the PCR method is 0.05%.

The comparison result of two kinds of methods shows, the detection sensitivity of test kit of the present invention and detection method thereof can reach 0.01%, and the detection sensitivity of PCR method is 0.05%; Through comparison, the sensitivity of test kit of the present invention and detection method is obvious Higher than the PCR method, can detect lower content of the sample.

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.

<110> Jilin Academy of Agricultural Sciences

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<120> LAMP detection primer set, kit and detection method for cry3A gene in transgenic plants

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Claims (7)

1. The LAMP detection primer set of cry3A gene in transgenic plants, including outer primer 1, outer primer 2, inner primer 1, inner primer 2 and loop primer, the nucleotide sequences of which are as follows: Outer primer 1: AAGCCCCACCTGTTCGA (SEQ ID NO: 1); Outer primer 2: GGCTCGCTGCTCTTGTTG (SEQ ID NO: 2); Internal primer 1: AGTTGAAGCTGTCGTTGCCGTACCTGCACCGCATCCAGTTC (SEQ ID NO: 3); Internal primer 2: GGAGCGGCAACTACGTGAGCAGAAGGGGCTGGTGATGA (SEQ ID NO: 4); Loop primer: GGGCTGGAAACGCGTGT (SEQ ID NO: 5). 2. A LAMP detection kit for the cry3A gene in transgenic plants, comprising the following components: (1) Detection primer solution: the detection primer solution prepared by 2 outer primers and 2 inner primers according to claim 1, the concentration of outer primer 1, outer primer 2, inner primer 1 and inner primer 2 is 4~ 6 μmol/L, 4~6 μmol/L, 32~48 μmol/L and 32~48 μmol/L; (2) Loop primer solution: prepared from the loop primer according to claim 1, the concentration of the loop primer is 16-24 μmol/L; (3) DNA polymerase with strand displacement activity: the concentration is 7~9 U/μL; (4) 10× reaction buffer: 200 mmol/L Tris-HCl, pH 8.8, 100 mmol/L KCl, 100 mmol/L (NH ₄ ) ₂ SO ₄ , 40~100 mmol/L MgSO ₄ , 6~14 mol/L betaine; (5) 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; (6) Positive DNA control samples, genomic DNA of cry3A transgenic plants, or Escherichia coli plasmid DNA containing cry3A gene sequence; (7) Negative DNA control sample: DNA without cry3A gene sequence; (8) Chromogen: 1000×SYBR GREEN I fluorescent dye. 3. The LAMP detection kit of cry3A gene according to claim 2, characterized in that: said detection primer solution contains 5 μmol/L outer primer 1, 5 μmol/L outer primer 2, 40 μmol/L inner Primer 1, 40 μmol/L internal primer 2. 4. The LAMP detection kit for cry3A gene according to claim 2, characterized in that: the loop primer solution contains 20 μmol/L loop primer. 5. The LAMP detection kit for cry3A gene according to claim 2, characterized in that: said DNA polymerase with strand displacement activity is Bst DNA polymerase at a concentration of 8 U/μL. 6 . The LAMP detection kit for cry3A gene according to claim 2 , wherein the concentrations of MgSO ₄ and betaine in the 10× reaction buffer are 80 mmol/L and 8 mol/L respectively. 7. utilize the method for detecting cry3A gene of the kit described in any one of claim 2～6, comprise 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, 1.5 μL of detection primer solution, 1 μL of loop primer solution, 1 μL of Bst DNA polymerase, and 10× reaction buffer into a 200 μL PCR reaction tube solution 2.5 μL, dNTPs solution 3 μL, make up to 25 μL with sterilized deionized water; (3) Prepare the LAMP detection reaction system of the control sample: When preparing the positive control and negative control reaction systems, except that the template DNA is replaced with the positive DNA control sample and the negative DNA control sample, the other components are consistent with step (2); (4) Run the LAMP amplification reaction: incubate at 63-65°C for 30-60 minutes, incubate at 80°C for 5 minutes to terminate the reaction; (5) Identification of LAMP amplification results: Judging the amplification results by visual observation or using a turbidimeter to observe the turbidity change of the precipitate in the reaction tube; or take 2~5 μL of the amplification product for agarose gel electrophoresis to detect the amplification Result; or add 1~2μL chromogen to the reaction tube, and judge the amplification result by visually observing the color development result.