Plant leaf DNA rapid extraction kit and application thereof

Abstract

The invention provides a plant leaf DNA rapid extraction kit and application thereof, the plant leaf DNA rapid extraction kit is used for rapidly extracting plant leaf DNA, and the kit comprises a DNA rapid extraction solution, a steel ball and a 96 deep hole plate. The rapid DNA extracting solution comprises 15-25 mmol / L of Tris-HCl, 20-30 mmol / L of NaCl, 0.4-0.6 mmol / L of EDTA (Ethylene Diamine Tetraacetic Acid) and 0.10-0.15% of SDS (Sodium Dodecyl Sulfate), and the pH value is 7.5-8.5. When the rapid DNA extracting solution is used, plant leaves, the rapid DNA extracting solution and steel balls are put into a 96 deep-well plate, are subjected to vibration grinding at 50-70 Hz for 30-70 seconds and are centrifuged at 3500-4500 r / min for 5-15 minutes, and supernatant is taken, namely the rapidly extracted plant DNA. The method is simple and rapid, high-temperature or organic solvent treatment is not needed, plant samples can be treated in a high-throughput mode, the concentration of the extracted DNA can reach 29.8-36.9 ng / mu L, and the extracted DNA is suitable for high-throughput genetic typing technologies such as fluorescent quantitative PCR, fluorescence labeled capillary electrophoresis multiple PCR detection and KASP, and has wide application prospects in the fields of crop variety detection, molecular assisted breeding and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA extraction, in particular to a rapid plant leaf DNA extraction kit and application thereof. Background Art

[0002] DNA extraction is fundamental and crucial for variety purity identification, requiring testing of a large number of individual strains. The extracted DNA concentration, homogenization, and extraction rate all significantly impact downstream experiments. DNA extraction methods include magnetic beads, a modified SDS method, CTAB, commercial kits, high-salt, low-pH methods, SDS proteinase K, alkaline boiling, and K-mate.

[0003] Currently, the most widely used nucleic acid extraction methods include magnetic beads, CTAB, modified SDS, and commercial kits. However, these methods are cumbersome, time-consuming, labor-intensive, and costly. While the high-salt, low-pH method, SDS proteinase K method, alkaline boiling, and K-mate methods are significantly faster, their extraction results are extremely unstable and have yet to be widely adopted. Rapid extraction methods such as cotton swabs and filter paper cannot achieve high-throughput sample extraction. While the alkaline boiling method, which is preferred for purity testing, can increase throughput and speed, the pH of the reagents used is easily affected by the environment, making it difficult to obtain high-quality DNA. The extraction quality varies between batches, making it unsuitable for high-throughput, rapid sample purity testing. Summary of the Invention

[0004] The present invention provides a plant leaf DNA rapid extraction kit and its application, which are used to solve the defects of low efficiency of conventional extraction methods and unstable extraction effect of rapid extraction methods in the prior art, and realize rapid, efficient and stable extraction of plant DNA.

[0005] In a first aspect, the present invention provides a rapid DNA extraction solution comprising the following components: 15-25 mmol / L Tris-HCl, 20-30 mmol / L NaCl, 0.4-0.6 mmol / L EDTA, 0.10-0.15% SDS, pH 7.5-8.5.

[0006] Preferably, the rapid DNA extraction solution comprises the following ingredients: 17-23 mmol / L Tris-HCl, 23-27 mmol / L NaCl, 0.45-0.55 mmol / L EDTA, 0.12-0.13% SDS, pH 7.8-8.2; More preferably, the rapid DNA extraction solution comprises the following ingredients: 20 mmol / L Tris-HCl, 25 mmol / L NaCl, 0.5 mmol / L EDTA, 0.125% SDS, pH 8.0.

[0007] Traditional alkaline digestion methods use NaOH to directly lyse cells, but this results in unstable DNA quality and significant pH fluctuations, which can affect PCR amplification. The rapid extraction solution of the present invention utilizes mild chemical lysis components that effectively disrupt plant cell walls and membranes while maintaining DNA integrity. By employing a buffer system with specific concentrations (Tris-HCl, EDTA, and salt ion control), DNA stability and high concentration are maintained, improving PCR amplification efficiency. Qubit assay analysis shows that the extracted DNA concentration is approximately 29.8–36.9 ng / μL, which is higher than the alkaline digestion method (0.2–0.9 ng / μL). Furthermore, this method provides enhanced DNA stability.

[0008] Furthermore, traditional commercial kits (such as those from QIAGEN and Tiangen) rely on silica columns and specialized buffers, resulting in a high cost of 5-10 yuan per sample, making them unsuitable for large-scale applications. The patented extraction solution utilizes conventional reagents (such as Tris, EDTA, NaCl, and low-concentration SDS), resulting in a low production cost, reducing the cost of extraction per sample to 0.1-0.5 yuan, an 80-90% reduction compared to commercial kits. The reagent formula is stable and can be stored for long periods, making it suitable for large-scale testing applications.

[0009] In a second aspect, the present invention provides a kit comprising the aforementioned rapid DNA extraction solution.

[0010] The present invention further provides the use of the above-mentioned rapid DNA extraction solution or the above-mentioned kit in DNA extraction.

[0011] In a third aspect, the present invention provides a method for extracting DNA, comprising the following steps: (1) Place the sample to be tested in a 96-well plate, add one steel bead and the above-mentioned DNA rapid extraction solution; (2) Centrifugation after vibration grinding; (3) The supernatant from centrifugation is the extracted sample DNA.

[0012] Preferably, the amount of the sample to be tested in step (1) of the above DNA extraction method is 3-6 mg of leaves.

[0013] Preferably, the diameter of the steel column in step (1) of the above DNA extraction method is 1.5-2.5 mm and the material is stainless steel.

[0014] Preferably, in the above-mentioned DNA extraction method, the oscillation frequency in step (2) is 50-70 Hz, and the time is 30-70 s.

[0015] Preferably, in the above DNA extraction method, the centrifugal speed in step (2) is 3500-4500 r / min, and the time is 5-15 min.

[0016] Preferably, the sample to be tested in step (1) of the above DNA extraction method is a plant leaf, preferably a plant seedling leaf.

[0017] Preferably, the plant is corn.

[0018] Existing CTAB, SDS, and commercial kit methods all require heating lysis, multiple centrifugation steps or chloroform extraction steps, resulting in a long DNA extraction time, usually 1 to 2 hours. The present invention adopts the "room temperature grinding method" to optimize the chemical composition of the extract and the mechanical grinding method, and directly performs mechanical grinding + liquid lysis in a 96-well plate to release DNA, which can then be directly used for PCR amplification without the need for high-temperature lysis, low-temperature treatment or organic solvent treatment and DNA precipitation or cleaning. The single-person 96-well DNA extraction speed is shortened to 15 minutes, which is 4-8 times faster than the traditional method; it avoids the cumbersome centrifugation and precipitation steps of the traditional method. It is suitable for seed companies, seed industry supervision or testing agencies to conduct large-scale purity testing.

[0019] The improved room-temperature grinding method established in this study is rapid, stable, and suitable for multiplex testing. It is suitable for high-throughput identification of crop variety purity. It particularly addresses the current problem of insufficient detection speed due to the annual increase in approved corn varieties and the corresponding increase in the number of tests. Based on a 96-well plate, a single person only needs about 15 minutes for extraction, which saves approximately 80 minutes compared to conventional commercial kit methods and still saves 10 minutes compared to the rapid alkaline boiling method. It also does not require high-temperature lysis, reducing the number of steps and improving the extraction efficiency of large-scale samples. Existing rapid high-throughput extraction methods, such as the alkaline boiling method, have low DNA extraction concentrations, which often requires increasing the sample volume in application, but also increases impurities and inhibits amplification.

[0020] The beneficial effects of the present invention include: The plant leaf DNA rapid extraction kit and its application provided by the present invention significantly improve the DNA extraction speed by optimizing the DNA extraction solution components and DNA extraction steps. The DNA extraction speed of a single 96-well plate is only 15 minutes, and the extracted DNA concentration reaches 29.8-36.9 ng / μL. The impurities are low, and the 8-plex amplification effect is stable. It is suitable for various application scenarios requiring high-throughput DNA extraction.

[0021] DNA extracted using the optimized room-temperature grinding method in this study showed bright bands in agarose gel electrophoresis, significantly improving extraction quality and concentration compared to rapid extraction methods such as the alkaline boiling method. The extraction reagents used in the room-temperature grinding method are stable, safe, and inexpensive conventional reagents. Compared to the filter paper method, the manual filter paper dipping and washing step is eliminated, saving labor costs and simplifying the extraction process. This allows for rapid and high-throughput DNA extraction, resolving the speed-limiting challenge of DNA extraction when testing large numbers of samples. This method has been successfully applied to crops such as corn and tomatoes, demonstrating excellent extraction results across a wide range of crops. The room-temperature grinding method currently enables rapid DNA extraction from crop leaves and etiolated seedlings, with relatively stable extraction quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Figure 1 shows the results of DNA fluorescence capillary electrophoresis using the rapid extraction reagents of various embodiments of the present invention and comparative examples. The ordinate represents peak height, the abscissa represents primers with different amplification ranges or fluorescence colors, and the numbers in the center of the graph represent amplification peaks at different sites. A: Example 1; B: Example 2; C: Example 3; D: Example 4.

[0024] Figure 2 Figure 2 shows the results of DNA fluorescence capillary electrophoresis using the rapid extraction reagents of various embodiments and comparative examples of the present invention. The ordinate represents peak height, the abscissa represents primers with different amplification ranges or fluorescence colors, and the numbers in the center of the graph represent amplification peaks at different sites. E: Example 5; F: Example 6; G: Comparative Example 1; H: Comparative Example 2.

[0025] Figure 3 Figure 1 shows the results of fluorescent capillary electrophoresis of DNA extracted using the rapid extraction reagents of various embodiments and comparative examples of the present invention. The ordinate represents peak height, the abscissa represents primers with different amplification ranges or fluorescence colors, and the numbers in the center of the graph represent amplification peaks at different sites. I: Comparative Example 3; J: Comparative Example 4; K: Comparative Example 5; L: Blank control.

[0026] Figure 4 The figure is a graph showing the agarose gel electrophoresis results of DNA extracted by the room temperature grinding method of the present invention and the conventional alkali boiling method, wherein lanes 1 to 4 are DNA extracted by the room temperature grinding method; lanes 5 to 8 are DNA extracted by the alkali boiling method.

[0027] Figure 5 The figure shows the fluorescent-labeled capillary electrophoresis results of eight-fold PCR of DNA extracted using the room-temperature grinding method of the present invention and the conventional alkali boiling method. A is the Jingke 968 DNA extracted by the room-temperature grinding method; B is the Jing724 DNA extracted by the room-temperature grinding method; C is the Jingke 968 DNA extracted by the alkali boiling method; and D is the Jing724 DNA extracted by the alkali boiling method. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1 DNA rapid extraction verification This embodiment provides a formula for a rapid DNA extraction solution, comprising the following ingredients: 15 mmol / L Tris-HCl, 20 mmol / L NaCl, 0.4 mmol / L EDTA, 0.075% SDS, pH 8.0.

[0030] Maize seeds of varieties Jingke 968 and Jing 724 (provided by the Beijing Academy of Agricultural and Forestry Sciences) were used for grinding using a HODER GENO 2010-230 and for centrifugation using a Beckman Avanti J-26S XP floor-standing high-efficiency centrifuge. Maize seeds were incubated in moist sand in the dark for 4 days until yellow coleoptiles developed.

[0031] The steps for rapid DNA extraction are as follows: (1) Place 5 mg of corn seedlings in a 96-well plate, add a 2 mm steel ball and 500 μL of DNA rapid extraction solution to each well, and seal the plate. (2) Grinding at 60 Hz for 50 s, followed by centrifugation at 4000 rpm for 10 min; (3) Take 80 μL of the centrifugal supernatant and store it at 4°C.

[0032] Example 2 The only difference between this embodiment and embodiment 1 is that the formula of the rapid DNA extraction solution includes the following ingredients: 16 mmol / L Tris-HCl, 23 mmol / L NaCl, 0.45 mmol / L EDTA, 0.1% SDS, pH 8.0.

[0033] Example 3 The only difference between this embodiment and embodiment 1 is that the formula of the rapid DNA extraction solution includes the following ingredients: 20 mmol / L Tris-HCl, 25 mmol / L NaCl, 0.5 mmol / L EDTA, 0.125% SDS, pH 8.0.

[0034] Example 4 The only difference between this embodiment and embodiment 1 is that the formula of the rapid DNA extraction solution includes the following ingredients: 22 mmol / L Tris-HCl, 28 mmol / L NaCl, 0.6 mmol / L EDTA, 0.15% SDS, pH 8.0.

[0035] Example 5 The only difference between this example and Example 3 is that the formula of the rapid DNA extraction solution includes the following ingredients: 25 mmol / L Tris-HCl, 30 mmol / L NaCl, 0.7 mmol / L EDTA, 0.175% SDS, pH 8.0.

[0036] Example 6 The only difference between this example and Example 3 is that the formula of the rapid DNA extraction solution includes the following ingredients: 27.5 mmol / L Tris-HCl, 32.5 mmol / L NaCl, 0.75 mmol / L EDTA, 0.2% SDS, pH 8.0.

[0037] Comparative Example 1 The only difference between this comparative example and Example 3 is that the formula of the DNA rapid extraction solution includes the following ingredients: 10 mmol / L Tris-HCl, 12.5 mmol / L NaCl, 1.25 mmol / L EDTA, 0.025% SDS, pH 8.0.

[0038] Comparative Example 2 The only difference between this comparative example and Example 3 is that the formula of the rapid DNA extraction solution includes the following ingredients: 30 mmol / L Tris-HCl, 50 mmol / L NaCl, 1 mmol / L EDTA, 0.25% SDS, pH 8.0.

[0039] Comparative Example 3 The only difference between this comparative example and Example 3 is that the 96-well plate was placed in an ice box for 30 minutes before vibration grinding.

[0040] Comparative Example 4 The only difference between this comparative example and Example 3 is that the 96-well plate was placed in a boiling water bath for 30 minutes before vibration grinding.

[0041] Comparative Example 5 The only difference between this comparative example and Example 3 is that the 96-well plate is replaced with a 2 mL centrifuge tube.

[0042] Example 5 DNA rapid extraction solution effect detection This example provides verification of the amplification effect of DNA extracted using the aforementioned rapid DNA extraction solution.

[0043] The corn DNA extracted from the above examples and comparative examples was mixed and amplified with nine primers (primer information is derived from the national standard GBT39914-2021), and the amplified products were detected by fluorescent capillary electrophoresis. The results are as follows: Figure 1 、 2 As shown in 3, the vertical axis in the figure is the peak height value, the horizontal axis is the primers with different amplification ranges or fluorescent colors, and the numbers in the center of the figure are the amplification peaks of different sites.

[0044] Depend on Figure 1 、 Figure 2 、 Figure 3 The results show that the DNA extracted using the schemes of Example 1, Example 2, Example 5, Example 6 and Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 all failed to amplify or only a small number of primers were successfully amplified. Although most of the primers were amplified in Example 4 and Comparative Example 5, the amplification peak was too low to meet the requirements of corn purity detection. All primers in Example 3 were successfully amplified and the peak was higher.

[0045] Example 6 Comparison of the rapid DNA extraction technology of the present invention with existing methods This example provides a comparison of the effectiveness of the aforementioned rapid DNA extraction technique (room temperature grinding) with existing DNA extraction methods. Maize seeds Jingke 968 and Jing 724 (provided by the Beijing Academy of Agriculture and Forestry Sciences) were used. Grinding was performed using a Hoder, GENO 2010-230, and centrifugation was performed using a Beckman, Avanti J.

[0046] The DNA rapid extraction solution used included the following components: 20 mmol / L Tris-HCl, 25 mmol / L NaCl, 0.5 mmol / L EDTA, 0.125% SDS, pH 8.0.

[0047] The extraction method is the same as that in Example 5, and the extraction time is less than 15 min.

[0048] The existing technology used for comparison is the alkali boiling method, which is also a rapid extraction method. The test method can be found in the national standard GBT39914-2021.

[0049] The DNA fragment size and impurity content of the supernatant were detected by agarose gel electrophoresis. Figure 4shown.

[0050] Depend on Figure 4 As shown, DNA extracted from corn leaves using the room-temperature grinding method showed clear bands (lanes 1-4) on agarose gel electrophoresis, with low levels of impurities such as protein within the pores and no obvious white streaking. However, DNA extracted using the alkaline boiling method showed no bands (lanes 5-8).

[0051] The DNA concentration of the supernatant was detected by a fluorescence quantitative instrument (Thermofisher Qubit 4.0). The results are shown in Table 1.

[0052] Table 1 Comparison of Qubit assay concentrations using different extraction methods As shown in Table 1, the DNA concentration of corn leaves extracted by the room temperature grinding method was between 29.8 and 36.9 ng / μL, with good uniformity and the DNA concentration could meet the requirements of downstream experiments such as PCR amplification. However, the DNA concentration of corn leaves extracted by the alkaline boiling method was only 0.2 to 0.9 ng / μL.

[0053] Eight specific primers for Jingke 968 and Jing 724 were used to perform octaplex PCR amplification on the DNA samples. The primer information was derived from the group standard "Maize Variety Purity Identification: InDel Molecular Marker Method" (T / CROPSSC 002-2023) issued by the Chinese Society of Crop Science. The results of fluorescence-labeled capillary electrophoresis are shown in the following table. Figure 5 shown.

[0054] Depend on Figure 5 It can be seen that the eight pairs of primers in the room temperature grinding method were successfully amplified, and the peak height was ideal ( Figure 5 A and B), while the alkaline boiling DNA octuplet PCR failed, and only two sites amplified fragments consistent with the expectations ( Figure 5 C, D).

[0055] 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 make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A rapid DNA extraction solution, characterized in that: Includes the following ingredients: 15-25mmol / L Tris-HCl, 20-30mmol / L NaCl, 0.4-0.6mmol / L EDTA, 0.10-0.15% SDS, pH 7.5-8.

5.

2. The rapid DNA extraction solution according to claim 1, characterized in that Includes the following ingredients: 17-23mmol / L Tris-HCl, 23-27mmol / L NaCl, 0.45-0.55mmol / L EDTA, 0.12-0.13% SDS, pH 7.8-8.2; Preferably, it comprises the following ingredients: 20 mmol / L Tris-HCl, 25 mmol / L NaCl, 0.5 mmol / L EDTA, 0.125% SDS, pH 8.

0.

3. A kit, characterized in that The invention comprises the rapid DNA extraction solution according to any one of claims 1 or 2.

4. Use of the rapid DNA extraction solution according to any one of claims 1 or 2 or the kit according to claim 3 in DNA extraction.

5. A DNA extraction method, characterized in that: The following steps are involved: (1) Place the sample to be tested in a 96-deep-well plate, add one steel ball and the DNA rapid extraction solution according to any one of claims 1 or 2; (2) Centrifugation after vibration grinding; (3) The supernatant from centrifugation is the extracted sample DNA.

6. The method according to claim 5, characterized in that The amount of the sample to be tested in step (1) is 3-6 mg of leaves.

7. The method according to any one of claims 5 or 6, characterized in that The steel ball in step (1) has a diameter of 1.5-2.5 mm and is made of stainless steel.

8. The method according to any one of claims 5 to 7, characterized in that: The oscillation frequency in step (2) is 50-70 Hz, and the time is 30-70 s.

9. The method according to any one of claims 5 to 8, characterized in that: The centrifugal speed in step (2) is 3500-4500 r / min, and the time is 5-15 min.

10. The method according to any one of claims 5 to 9, characterized in that: The sample to be tested in step (1) is a plant leaf, preferably a plant seedling leaf.

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