Method for detecting CRISPR (clustered regularly interspaced short palindromic repeats) related Cas9 gene in gene editing plant based on LAMP-HNB and application
Through the detection method based on LAMP-HNB, the problem of Cas9 sequence detection in genome-edited plants is solved, and a fast, simple and specific detection effect is achieved, which is suitable for on-site applications.
Patent Information
- Application Number
- CN202411580771.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-07
- Publication Date
- 2025-03-28
AI Technical Summary
It is difficult to develop a fast, simple and highly specific analytical method to qualitatively detect Cas9 sequences in genome-edited plants.
Using a detection method based on LAMP-HNB, DNA was extracted, PCR amplification targeting the Cas9 coding sequence was performed, and F3 and B3, LF and LB, FIP and BIP were used as primers in the LAMP-HNB reaction, and HNB was added as color indicators. The color of the reaction solution was observed to determine whether the target gene was found in the sample to be tested.
The method is faster, more cost-effective, no thermal cycler required, suitable for on-site use, with higher sensitivity and specificity, and makes the results interpreted simply and clearly through visual readings of HNB.
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Figure CN119709959A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technology and relates to a detection method and application of CRISPR-related Cas9 genes in gene-edited plants based on LAMP-HNB. Background Art
[0002] The development and implementation of genome editing technologies have revolutionized the field of genetic engineering, especially with the advent of CRISPR / Cas9. Known for its precision and reliability, this system allows for targeted modifications within the plant genome, offering potential solutions for agricultural advancement and a variety of biological research applications. Despite its potential, CRISPR / Cas9 technology raises significant biosafety and ethical issues, particularly the risk of off-target effects due to the continuous activity of the CRISPR system in organisms. These off-target modifications can lead to unintended genetic changes, which raise safety concerns and require the development of reliable detection methods.
[0003] Typically, the CRISPR / Cas machinery for genomic insertion is isolated during the breeding process for genome-edited crops. However, if the CRISPR / Cas9 construct or the Cas9 coding sequence remains in the plant genome; current methods can detect Cas9 applications. Strategies targeting common elements, such as the CaMV P-35S promoter and the T-nos terminator, have been published. While other techniques focus on detecting specific DNA modifications at the nucleotide level, which requires prior knowledge of the target sequence. PCR and qPCR methods for detecting the full class of genome editing products, such as methods targeting Cas9 and Cas12 (cpf1) coding sequences in gene-edited plant organisms, are under development or have been implemented. However, to our knowledge, no isothermal amplification methods targeting Cas9 coding sequences have been published so far. Summary of the invention
[0004] The present invention provides a method for detecting CRISPR-related Cas9 genes in gene-edited plants based on LAMP-HNB, so as to solve the problem that it is difficult to develop a fast, simple and highly specific analysis method for qualitatively detecting Cas9 sequences in genome-edited plants using the existing technology.
[0005] The present invention adopts the following technical solution:
[0006] The detection method of CRISPR-related Cas9 genes in gene-edited plants based on LAMP-HNB includes the following steps:
[0007] (1) Extract DNA from the sample to be tested;
[0008] (2) Using DNA as a template, PCR amplification of the target fragment was performed using primers F3 and B3 targeting the Cas9 coding sequence;
[0009] (3) Using the obtained target fragment as a template, F3 and B3, LF and LB, and FIP and BIP as primers, and adding HNB at the same time, perform a LAMP-HNB reaction, observe the color of the reaction solution, and determine whether the sample to be tested contains the target gene based on the color.
[0010] The use of LAMP in conjunction with HNB offers several significant advantages for detecting genome-edited plants. LAMP is known for its rapid amplification capabilities, generating billions of copies of target DNA within an hour under isothermal conditions, eliminating the need for thermal cycling and thus simplifying the process. This high efficiency is complemented by the specificity of LAMP, which ensures precise targeting of the desired DNA sequence through the use of multiple primers. The incorporation of HNB as a colorimetric indicator further enhances the practicality of this method. HNB allows for direct visual detection, with a clear color change from purple to sky blue indicating a positive result. This visual readout does not require complex equipment, making the analysis easily accessible and interpretable in a variety of settings. Due to the advantages mentioned above, the present invention has developed a successful LAMP-HNB system to detect Cas9 coding sequences in Cas9 gene-edited plants.
[0011] Preferably, the sequence of primer FIP is shown as SEQ ID NO.1, and the sequence of primer BIP is shown as SEQ ID NO.2.
[0012] Preferably, the sequence of primer F3 is shown as SEQ ID NO.3, and the sequence of primer B3 is shown as SEQ ID NO.4.
[0013] Preferably, the sequence of primer LF is shown as SEQ ID NO.5, and the sequence of primer LB is shown as SEQ ID NO.6.
[0014] Preferably, the reaction mixture of the LAMP-HNB reaction contains Tris-HCl, KCl, (NH4)2SO4, Triton X-100, dNTP and MgSO4, FIP primer, BIP primer, F3 primer, B3 primer, LF primer, LB primer, template, BstDNA polymerase, and HNB dye.
[0015] Preferably, the concentration of MgSO4 in the reaction mixture is 6 mM.
[0016] Preferably, the added amount of the HNB dye is 120 μM.
[0017] Preferably, the amount of Bst DNA polymerase added to the reaction mixture is 0.32 U / μL.
[0018] Further preferably, the reaction mixture of the LAMP-HNB reaction contains 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 0.1% Triton X-100, 1.4mM dNTP and 6mM MgSO4, 1.6mM FIP primer, 1.6mM BIP primer, 0.2mM F3 primer, 0.2mM B3 primer, 0.4mM LF primer, 0.4mM LB primer, 1μL template, 0.32U / μL Bst DNA polymerase, 120μM HNB dye, and ultrapure water is added to 25μL.
[0019] Preferably, the reaction temperature of the LAMP-HNB reaction is 65°C.
[0020] Preferably, the reaction time of the LAMP-HNB reaction is 30 min.
[0021] The present invention also provides an application of the above detection method for detecting CRISPR-related Cas9 genes in gene-edited tomatoes.
[0022] By implementing the above technical solution, the present invention has the following beneficial effects:
[0023] The detection method provided by the present invention is faster and more cost-effective than PCR, and does not require a thermal cycler, making it very suitable for field use. Compared with other isothermal methods, LAMP has higher sensitivity and specificity, and combined with HNB, the results can be easily and intuitively interpreted without the need for additional equipment. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The results of PCR electrophoresis of the Cas9 coding sequence in the targeted genome editing vector pYLCRISPR / Cas9P35s-N using agarose gel are shown. Wherein, M: Sangon Biotech DNA marker (100-2000bp); 1. Genome editing vector pYLCRISPR / Cas9P35s-N; 2. Genome editing vector pYLCRISPR / Cas9P35s-N; 3. Negative control (ddH2O). Expected band size: 189bp.
[0025] Figure 2Agarose gel electrophoresis showing the results of PCR targeting Cas9 coding sequences in genome-edited plants. M: Sangon Biotech DNA marker (100-2000 bp); 1. Genome editing vector pYLCRISPR / Cas9P35s-N (positive control); 2. SPB8a 4th; 3. WRKY1 6th; 4. WRKY1 7th; 5. GR2 3rd; 6. Wild-type tomato; 7. Negative control (ddH2O). Expected band size: 189 bp.
[0026] Figure 3 Results of LAMP without HNB using agarose gel electrophoresis and visual observation are shown. Positive samples show a ladder of multiple bands of different sizes in agarose gel (A), while the naked eye is expected to be turbid due to precipitation of magnesium pyrophosphate (B). M: Sangon Biotech DNA marker (100-2000bp); 1. Genome editing vector pYLCRISPR / Cas9P35s-N (positive control); 2. Genome editing vector pYLCRISPR / Cas9P35s-N (positive control); 3. Negative control (ddH2O).
[0027] Figure 4 The specific results of electrophoresis and naked eye are shown. M: Sangon Biotech DNA marker (100-2000bp); 1. Cas9 gene-edited tomato; 2. Wild-type tomato; 3. Transgenic Arabidopsis; 4. Transgenic corn; 5. Transgenic strawberry; 6. Cas12 gene-edited corn; 7. Negative control (ddH2O).
[0028] Figure 5 The sensitivity results of electrophoresis and naked eye are shown. M: Sangon Biotech DNA marker (100-2000bp); 1.4x10 8 Quantity (inventory); 2.10 8 3.10 7 4.10 6 5.10 5 6.10 4 7.10 3 100 parts; 8. 100 parts; 9. 10 parts; 10. 1 parts; 11. Negative control (ddH2O).
[0029] Figure 6The applicability of LAMP detection obtained by electrophoresis and naked eye in Cas9 genome editing of tomatoes is shown. M: Sangon Biotech DNA marker (100-2000bp); 1. Positive control; 2. SPB8a 4th; 3. WRKY1 6th; 4. WRKY1 7th; 5. GR2 3rd; 6. Wild-type tomato; 7. Negative control (ddH2O). DETAILED DESCRIPTION
[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0031] The experimental methods for which specific conditions are not indicated in the following examples are usually performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or under conditions recommended by the manufacturer.
[0032] All primer sequences were synthesized and purified by Tsingke Biotechnology (Shanghai, China). SanPrep column plasmid miniprep kit, 2X SanTaq PCR mix, nuclease-free water, and DNA markers were purchased from Sangon Biotech, Shanghai, Switzerland. DNTP and kanamycin (CAS: 25389-94-0) were purchased from Bio Basic, Shanghai, Switzerland. Bst DNA polymerase, MgSO4, and LAMP buffer were purchased from New England Biolabs, Hitchin, UK. Hydroxynaphthol blue (HNB) (CAS: 63451-35-4) was purchased from Sigma-Aldrich, St. Louis, MO, USA. An Escherichia coli strain and pYLCRISPR / Cas9P35s-N vector (size: 15011 bp, containing the Cas9 coding sequence) and four DNA samples from the CRISPR / Cas9 genome-edited plant tomato (named SPB8a 4 and SPB8b 5 ) were obtained from Hefei University of Technology. th 、WRKY16 th 、WRKY1 7 th and GR2 3 rd) and wild-type tomato (non-gene-edited) DNA. Cas12 gene-edited maize samples, transgenic samples (Arabidopsis, maize, and strawberry) were from Shanghai University, China, and Shanghai Academy of Agricultural Sciences, China, respectively.
[0033] Example 1 Extraction of plasmid DNA
[0034] The pYLCRISPR / Cas9P35s-N vector (Gene Bank: KR029112.1) containing Escherichia coli previously stored in a magnetic bead cryovial at -80°C was inoculated in a Luria Bertani (LB) + kanamycin (50 μg / mL) agar plate. After 24 hours of normal inverted culture, the bacteria were transferred to liquid LB + kanamycin (50 μg / mL) medium and shaken overnight. The glycerol stock of the bacterial culture was stored at -20°C. According to the instructions of the SanPrep chromatography column plasmid mini-prep kit, 5 mL of bacterial culture was used for plasmid extraction, and the resulting 100 μL elution sample was frozen at -20°C. Agarose gel electrophoresis was used to verify the plasmid size, and 4×10 8 The concentration of the copy plasmid DNA.
[0035] Example 2 Design of LAMP primers
[0036] A set of six LAMP primers were designed based on the optimized Cas9 coding gene sequence in the pYLCRISPR / Cas9P35s-N vector (SEQ ID NO.7) using PrimerExplorerV4 software (http: / / primerexplorer.jp / e / ), and the primer names and sequences correspond to SEQ ID NO.1 to 6. The specificity and Tm values of these primers were tested by the website tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ). Table 1 below lists the primers used in this study, and Table 2 shows the Cas9 targeting coding sequence used as a reference for primer design in the present invention.
[0037] Table 1. Oligonucleotide primers used in the present invention Primer DNA sequences (5'-3')
[0038]
[0039] Table 2. Cas9 DNA coding sequence used as a reference for primer design in the present invention Name DNA sequence (5'-3')
[0040]
[0041]
[0042] Example 3 PCR of plasmid samples and genome-edited plants
[0043] To verify the identity of the extracted plasmids and the presence of the Cas9 coding sequence in the four genome-edited plants (named SPB8a4th, WRKY1 6th, WRKY1 7th, and GR2 3rd), PCR was performed using the F3 and B3 primers targeting the Cas9 coding sequence. The 2X SanTaq PCR mix (which already contains Taq DNA polymerase, PCR buffer, and loading buffer) and its recommended PCR conditions were used according to the manufacturer's instructions. The use of F3 and B3 primers to verify their functionality, after amplification, the expected band size in the agarose gel was 189 bp.
[0044] We observed that amplification in samples using only the genome editing vector pYLCRISPR / Cas9P35s-N resulted in a band of approximately 189 bp, see Figure 1 The observed band size matched the expected size, confirming that the plasmid contained the Cas9 coding sequence and could be used as a positive control for subsequent experiments.
[0045] See also Figure 2 When tested in genome-edited plants, both the positive control and genome-edited plant samples produced the expected band of approximately 189 bp. In contrast, neither the wild-type nor the negative control samples showed any bands as expected because they do not contain the Cas9 coding sequence. These results confirmed that the genome-edited samples contained the Cas9 coding sequence, validating their suitability for evaluating LAMP-HNB reactions.
[0046] Example 4 LAMP-HNB reaction
[0047] The initial LAMP reaction was performed in the absence of HNB. The initial reaction was performed in a 25 μL reaction mixture containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% TritonX-100, 8 mM MgSO4, 1.4 mM DNTP, 1.6 mM FIP primer, 1.6 mM BIP primer, 0.2 mM F3 primer, 0.2 mM B3 primer, 0.4 mM LF primer, 0.4 mM LB primer, 1 μL template, 0.32 U / μL Bst DNA polymerase, 120 μM HNB dye. The initial reaction conditions were 40 minutes at 65°C. Positive control (see Figure 3Samples 1 and 2) produced a ladder containing multiple bands of different sizes in the agarose gel, and as expected, turbidity was visible to the naked eye due to magnesium pyrophosphate precipitation. The negative control showed no amplification or turbidity. The results indicate that the LAMP reaction was successful and optimization experiments can be started.
[0048] Optimization of LAMP-HNB reaction conditions
[0049] The LAMP-HNB detection conditions were optimized using a pYLCRISPR / Cas9P35s-N DNA plasmid (positive control) with a total volume of 25 μL. The reaction concentrations of the reagents were optimized within the following value ranges: HNB (120 and 150 μM), MgSO4 (4, 6, 8 and 10 mM) and Bst DNA polymerase (0.04, 0.08, 0.16 and 0.32 U / μL). The LAMP analysis was also optimized by testing different reaction temperatures (50, 55, 60, 65 and 70 ° C) for 30 minutes. At the optimal temperature, the reaction time (30, 45 and 60 minutes) was also evaluated. In order to select the optimal parameters, the analysis was evaluated based on the HNB visualized color change of the 2% LAMP product and / or electrophoresis in agarose gel. All experiments were performed in triplicate.
[0050] Optimization of HNB concentration: LAMP reaction was performed in a 25 μL reaction mixture containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 1.4 mM dNTP and 6 mM MgSO4, 1.6 mM FIP primer, 1.6 mM BIP primer, 0.2 mM F3 primer, 0.2 mM B3 primer, 0.4 mM LF primer, 0.4 mM LB primer, 1 μL template, 0.32 U / μL Bst DNA polymerase, HNB dye (120 μM, 150 μM), and ultrapure water was added to 25 μL. The reaction system was incubated at 65°C for 30 minutes in a Gene Explorer Touch thermal cycler (Bio-Gener Technology, Hangzhou, China).
[0051] Optimization of MgSO4 concentration: LAMP reaction was performed in a 25 μL reaction mixture containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 1.4 mM dNTP and MgSO4 (4, 6, 8 and 10 mM), 1.6 mM FIP primer, 1.6 mM BIP primer, 0.2 mM F3 primer, 0.2 mM B3 primer, 0.4 mM LF primer, 0.4 mM LB primer, 1 μL template, 0.32 U / μL Bst DNA polymerase, HNB dye (120 μM), and ultrapure water was added to 25 μL. The reaction system was incubated at 65°C for 30 minutes in a Gene Explorer Touch thermal cycler (Bio-Gener Technology, Hangzhou, China).
[0052] Optimization of Bst DNA polymerase addition: LAMP reaction was performed in a 25 μL reaction mixture containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 1.4 mM dNTP and 6 mM MgSO4, 1.6 mM FIP primer, 1.6 mM BIP primer, 0.2 mM F3 primer, 0.2 mM B3 primer, 0.4 mM LF primer, 0.4 mM LB primer, 1 μL template, Bst DNA polymerase (0.04, 0.08, 0.16 and 0.32 U / μL), HNB dye (120 μM), and ultrapure water was added to 25 μL. The reaction system was incubated at 65°C for 30 minutes in a Gene Explorer Touch thermal cycler (Bio-GenerTechnology, Hangzhou, China).
[0053] result:
[0054] Regarding the optimization of HNB concentration, two concentrations (120 and 150 μM) were tested. Both concentrations showed a clear color change from purple to blue, indicating successful amplification. However, analysis of agarose gels showed that the 120 μM concentration produced clearer bands. Therefore, the 120 μM concentration was used for subsequent experiments.
[0055] To optimize MgSO4, the bands in the agarose gel were clearer at concentrations of 4, 6, and 8 mM. However, when observed by naked eye, only the 6 mM concentration provided the best color contrast between purple and blue. Therefore, the 6 mM concentration was used for subsequent experiments.
[0056] Optimization experiments with Bst DNA polymerase showed that a concentration of only 0.32 U / μL resulted in a visible color change from purple to blue and clearer bands in agarose gels. Therefore, the following experiments used a Bst DNA polymerase concentration of 0.32 U / μL.
[0057] Optimized reaction system: LAMP reaction was carried out in a 25 μL reaction mixture containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Triton X-100, 1.4 mM dNTP and 6 mM MgSO4, 1.6 mM FIP primer, 1.6 mM BIP primer, 0.2 mM F3 primer, 0.2 mM B3 primer, 0.4 mM LF primer, 0.4 mM LB primer, 1 μL template, 0.32 U / μL Bst DNA polymerase, HNB dye (120 μM), and ultrapure water was added to 25 μL.
[0058] Optimization of reaction temperature: Under the optimized reaction system, the cells were incubated at different reaction temperatures (50, 55, 60, 65, and 70°C) for 30 min in a Gene Explorer Touch thermal cycler (Bio-Gener Technology, Hangzhou, China).
[0059] Optimization of reaction time: Under the optimized reaction system, the cells were incubated at 65°C for 30, 45, and 60 min in a Gene Explorer Touch thermal cycler (Bio-Gener Technology, Hangzhou, China).
[0060] Optimal reaction conditions were determined by electrophoresis in 2% agarose gel using an EPS-300 power source and band visualization using an AlphaImager Mini UVTransilluminator.
[0061] result:
[0062] Regarding temperature, at temperatures of 60°C and 65°C, good color changes and clear bands in agarose gel were obtained by naked eye; lower temperatures converted negative controls into positive controls, while higher temperatures failed to produce amplification. Therefore, a temperature of 65°C was selected for the following experiments.
[0063] Regarding time optimization, different times (30, 45, and 60 minutes) were tested. Both 30 and 45 minutes provided good amplification results, with clear bands in the agarose gel and sharp color contrast from purple to blue. However, at 60 minutes, the negative control began to show false positives. Considering the time efficiency and reaction performance, 30 minutes was selected for the following experiments.
[0064] Example 5 Specificity test of LAMP-HNB detection
[0065] DNA extracted from Cas9 gene-edited tomatoes, three other plant transgenic samples (Arabidopsis, corn, and strawberry), Cas12 gene-edited corn, and wild-type tomatoes were used as templates to react under the LAMP assay conditions optimized in Example 4. The results were analyzed by visualizing the products using 2% agarose gel electrophoresis and the color change of HNB dye with the naked eye.
[0066] The results show that Figure 4 ), only the Cas9 gene-edited tomato showed amplification, characterized by a ladder of multiple bands of different sizes visible in the agarose gel. In addition, a change from purple to blue was observed in the sample tube, which could be detected by the naked eye. In contrast, no amplification or color change was observed in the tubes of other samples, indicating that the established method for detecting Cas9 is highly specific.
[0067] Example 6 Sensitivity test of LAMP-HNB detection
[0068] The pYLCRISPR / Cas9P35s-N DNA vector was used as a sample and the detection limit was determined to determine the sensitivity of the LAMP assay. The sample was serially diluted 10-fold in nuclease-free water, ranging from 4 × 10 8 The cloned protein was amplified from 1 to 1 copy and used as a template under the optimized LAMP assay conditions of Example 4, and the product was then visualized by 2% agarose gel electrophoresis and HNB dye color change with the naked eye.
[0069] The results show that ( Figure 5 ), even at a concentration of 1 copy of the Cas9 targeting sequence, amplification can be detected as a band in the agarose gel and a color change from purple to blue.
[0070] Example 7 Determination of suitability of Cas9 genome editing in plants
[0071] To evaluate the suitability of LAMP for detecting the Cas9 DNA cassette in Cas9 genome-edited plants, a positive control (pYLCRISPR / Cas9P35s-N DNA vector), four Cas9 genome-edited tomatoes (SPB8a 4th, WRKY1 6th, WRKY1 7th, and GR2 3rd), a wild-type tomato (non-gene editing), and a negative control (LAMP mixture + 1 μL nuclease-free water) were analyzed. The products were reacted under the optimized LAMP assay conditions of Example 4, and the products were visualized by electrophoresis with 2% agarose gel and changing the HNB color of the samples from purple to blue. All experiments were tested in triplicate.
[0072] The results show that ( Figure 6 ), the technique can effectively detect various Cas9 genome-edited tomatoes modified with the same vector and distinguish them from negative samples (wild-type tomatoes and negative controls). Clear bands were observed in the agarose gel of genome-edited tomatoes, and all of these samples turned blue to the naked eye. In contrast, no amplification and retention of purple were observed in wild-type and negative control samples. These findings indicate that the technique can effectively detect Cas9 coding sequences in various Cas9 genome-edited tomatoes.
[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for detecting CRISPR-related Cas9 genes in gene-edited plants based on LAMP-HNB, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Using DNA as a template, PCR amplification of the target fragment was performed using primers F3 and B3 targeting the Cas9 coding sequence; (3) Using the obtained target fragment as a template, F3 and B3, LF and LB, and FIP and BIP as primers, and adding HNB at the same time, perform a LAMP-HNB reaction, observe the color of the reaction solution, and determine whether the sample to be tested contains the target gene based on the color.
2. The detection method according to claim 1, characterized in that: The sequence of primer FIP is shown as SEQ ID NO.1, and the sequence of primer BIP is shown as SEQ ID NO.
2.
3. The detection method according to claim 2, characterized in that: The sequence of primer F3 is shown in SEQ ID NO.3, and the sequence of primer B3 is shown in SEQ ID NO.
4.
4. The detection method according to claim 3, characterized in that: The sequence of primer LF is shown as SEQ ID NO.5, and the sequence of primer LB is shown as SEQ ID NO.
6.
5. The detection method according to claim 4, characterized in that: The reaction mixture of the LAMP-HNB reaction contains Tris-HCl, KCl, (NH4)2SO4, Triton X-100, dNTP and MgSO4, FIP primer, BIP primer, F3 primer, B3 primer, LF primer, LB primer, template, Bst DNA polymerase, and HNB dye.
6. The detection method according to claim 5, characterized in that: The amount of HNB dye added to the reaction mixture was 120 μM.
7. The detection method according to claim 5, characterized in that: The concentration of MgSO4 in the reaction mixture was 6 mM.
8. The detection method according to claim 5, characterized in that: The amount of Bst DNA polymerase added to the reaction mixture was 0.32 U / μL.
9. The detection method according to claim 5, characterized in that: The reaction mixture of the LAMP-HNB reaction contains 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH4)2SO4, 0.1% Triton X-100, 1.4mMdNTP and 6mM MgSO4, 1.6mM FIP primer, 1.6mM BIP primer, 0.2mM F3 primer, 0.2mM B3 primer, 0.4mM Lf primer, 0.4mM Lb primer, 1μL template, 0.32U / μL Bst DNA polymerase, HNB dye, and ultrapure water is added to 25μL.
10. The detection method according to claim 9, characterized in that: The reaction temperature of the LAMP-HNB reaction was 65°C.
11. The detection method according to claim 9, characterized in that: The reaction time of the LAMP-HNB reaction is 30 min.
12. Use of the detection method according to any one of claims 1 to 11, characterized in that: Used for detection of CRISPR-related Cas9 genes in gene-edited tomatoes.