Nucleic acid extracting solution suitable for plant leaves, rapid extracting method and application of nucleic acid extracting solution

Abstract

The invention relates to the technical field of molecular biology breeding, and particularly discloses a nucleic acid extracting solution suitable for plant leaves, a rapid extracting method and application of the nucleic acid extracting solution. The nucleic acid extracting solution is prepared from 60 mM of Tris-HCl, 20 mM of EDTA and 0.5% of SDS in mass / volume ratio. The nucleic acid extraction comprises the following specific operation steps: smashing plant leaves, adding a nucleic acid extracting solution into the powder, shaking and uniformly mixing, and performing high-temperature water bath to obtain the extracted nucleic acid. The nucleic acid extracting solution disclosed by the invention has the following advantages: 1) nucleic acid in plant leaves is quickly released for downstream experiments such as genotyping; 2) the nucleic acid extracting solution does not contain toxic reagents, so that the safety of experiment operators can be ensured; and 3) the extraction method does not need any purification step, so that the operation time is greatly shortened, and the method has the advantages of simplicity, convenience and rapidness in operation.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology breeding technology, in particular to a nucleic acid extraction solution suitable for plant leaves, a rapid extraction method and application thereof. BACKGROUND

[0002] Molecular breeding is a revolutionary technology in the field of modern agriculture and biotechnology. It combines molecular biology, genomics, bioinformatics and other modern techniques to precisely manipulate and optimize genetic information of plants and animals, achieving efficient and targeted genetic improvement. Compared with traditional breeding methods that rely on phenotype observation and natural crossing, molecular breeding can significantly shorten the breeding cycle, improve the accuracy of target traits, and break through the genetic barriers between species, providing key technical support for solving global problems such as food security, resource shortage and climate change. The core technology of molecular breeding includes marker-assisted selection (MAS), which can select excellent individuals with target traits through the linkage relationship between DNA markers and target traits. DNA marker identification is the key to screening individuals.

[0003] Advantages of high-throughput genotyping and fluorescence PCR application: DNA marker detection technology based on polymerase chain reaction (PCR) is the current mainstream technology. It is a molecular identification method used for exponential amplification of specific nucleic acid fragments and determination of the presence of DNA markers by fluorescence signal. Its greatest feature is that it can obtain a large number of identical fragments after cloning of trace amounts of nucleic acids, and perform quantitative detection according to the fluorescence signal of the fragments. The PCR process is an enzymatic reaction, and the enzyme catalysis requires relatively pure nucleic acid samples that meet the requirements.

[0004] Challenges of existing nucleic acid extraction methods: In current molecular detection, the commonly used extraction methods are magnetic bead method and column membrane method. These two methods can obtain relatively pure nucleic acid samples. Although the magnetic bead method can be adapted to an automated platform, both methods still require multiple steps for purification, and the cost of related automated equipment and consumables is high. At the same time, toxic reagents may be used in the extraction process, which can harm the health of the operator.

[0005] Therefore, it is still an urgent problem in molecular breeding to develop a non-toxic and harmless, simple, low-cost and easy-to-operate nucleic acid extraction method. SUMMARY

[0006] The present application aims to provide a nucleic acid extraction solution suitable for plant leaves, which can not only obtain sufficient DNA for subsequent PCR experiments, but also has simpler components without reducing the extraction effect of existing extraction methods.

[0007] In addition, the present application also provides a rapid extraction method based on the above nucleic acid extraction solution and its application, to reduce the use of toxic reagents in the nucleic acid extraction process, simplify the nucleic acid extraction steps, and greatly shorten the nucleic acid extraction and purification time, thereby significantly improving the detection throughput.

[0008] The present application is realized by the following technical solutions:

[0009] The nucleic acid extraction solution suitable for plant leaves is prepared from 60 mM Tris-HCl, 20 mM EDTA and 0.5% SDS (mass / volume ratio).

[0010] Based on the problems existing in the current nucleic acid extraction, and in order to reduce the use of toxic reagents in the nucleic acid extraction process, simplify the nucleic acid extraction steps, and shorten the nucleic acid extraction and purification time, the above nucleic acid extraction solution is designed.

[0011] The above nucleic acid extraction solution of the present application not only has a simple formula, but also obtains relatively stable DNA for plant leaf nucleic acid extraction, and sufficient DNA can be obtained for subsequent PCR experiments using a simple extraction solution. Compared with the existing extraction methods, the extraction effect is not reduced, and the components of the extraction solution are simpler.

[0012] Although the three components of the nucleic acid extraction solution of the present application are common reagents for nucleic acid extraction, before the present application, Tris-HCl, EDTA and SDS all need to be combined with other reagents for nucleic acid extraction, for example: CN117778378A discloses a nucleic acid extraction solution kit and its application, which combines lauryl maltose neopentyl glycol, Tween-20, and at least one of guanidine salt, buffer, sodium salt, ethylenediaminetetraacetic acid, and isopropyl alcohol for DNA extraction of viruses; CN118256486A discloses a nucleic acid extraction solution nucleic acid extraction kit and its application, which combines (5-cyclohexylpentyl)-beta-D-maltoside, Triton X-100 and guanidine salt, buffer, sodium dodecyl sulfate, dithiothreitol for DNA extraction of viruses; CN CN115521932B discloses a nucleic acid extraction solution system and kit, which combines guanidinium isothiocyanate, SDS, N-methyl acetamide, tetraethylene glycol; Triton X-100, Tris-HCl, EDTA·2Na, and silicon hydroxyl magnetic beads for DNA extraction of plasma samples.

[0013] However, the prior art does not provide a combination of Tris-HCl, EDTA and SDS at specific concentrations for nucleic acid extraction from plant leaves, which achieves the effect of simpler components of the extraction solution without reducing the extraction effect of the existing extraction method.

[0014] The effects of Tris-HCl, EDTA and SDS at specific concentrations in the acid extraction solution of the present application are as follows:

[0015] Tris-HCl (60mM): Construct a stable pH environment for DNA.

[0016] (1) pH buffering mechanism: Tris-HCl at a concentration of 60mM can maintain the pH of the system at 7.5-8.0 (the optimal stable interval for DNA). DNA strands are prone to depurination under acidic conditions and strand separation under alkaline conditions, and stable pH can prevent the breakage of nucleic acid backbone due to acid-base fluctuations.

[0017] (2) Concentration rationality: 60mM is a moderate strength buffer system that can resist the release of acidic substances (such as metabolites) during cell lysis, and will not affect subsequent enzyme reactions (such as PCR) due to excessive buffering capacity.

[0018] EDTA (20mM): High efficiency inhibition of nuclease activity.

[0019] Metal ion chelation: EDTA chelates the essential cofactors of Mg 2+ , Ca 2+ and other nucleases (such as DNase) at high concentrations (20mM), making the enzyme active center inactive. Compared with the conventional concentration of 1-5mM, 20mM EDTA has stronger inhibitory effect on stubborn nucleases (such as DNase in serum and tissue), which can effectively block the DNA degradation pathway.

[0020] Additional stabilization: EDTA can also bind to metal ions on the surface of DNA double strands, reducing ion-mediated DNA conformation changes and indirectly enhancing double strand stability.

[0021] SDS (0.5%): Double protection of DNA from enzymatic degradation.

[0022] Nuclease denaturation: 0.5% SDS as a strong detergent can destroy the protein structure (such as disulfide bond, hydrophobic group) of nucleases, making them completely inactive. This concentration can effectively denature intracellular nucleases, and avoid the difficulty of removing SDS due to high concentration (such as 1%), which in turn interferes with subsequent DNA operations (such as enzyme digestion, sequencing).

[0023] Synergistic effect of lysis and protection: SDS releases DNA by destroying cell membranes, and its negatively charged sulfate group can bind to DNA to form a "protective layer", reducing the probability of DNA contacting nucleases.

[0024] The method for rapidly extracting nucleic acid from plant leaves based on the nucleic acid extraction solution comprises the following steps:

[0025] After the plant leaves are crushed, the crushed powder is obtained, the nucleic acid extraction solution is added to the crushed powder, and the mixture is shaken and mixed and subjected to high-temperature water bath, so that the extracted nucleic acid is obtained.

[0026] The method for extracting nucleic acid from plant leaves based on the acid extraction solution has the following advantages:

[0027] 1) Rapid release of nucleic acid in plant leaves for downstream experiments such as genotyping;

[0028] 2) The nucleic acid extraction solution does not contain toxic reagents, which can ensure the safety of experimental operators;

[0029] 3) The extraction method does not go through any purification steps, greatly shortening the operation time, and has the advantages of simple operation and quickness.

[0030] In summary, the rapid extraction method of the application has the following advantages: simple workflow: using a 96-well plate, the extraction time of a single plate is only 15 minutes, and the extracted nucleic acid does not need to be subjected to concentration and purity detection.

[0031] Further, the sample types of plant leaves include fresh, dried plant seedling leaves and leaves obtained by tissue culture.

[0032] Further, the process of obtaining the crushed powder is as follows:

[0033] 0.4-1.6mg is sampled using a puncher, the obtained sample is placed in a 96-well deep well plate or a field sampling tube, and a 5mm diameter steel ball is used to crush.

[0034] Further, the temperature of the high-temperature water bath is 90℃, and the time is 10min.

[0035] Further, during the high-temperature water bath process, the mixture is shaken and mixed every 5min for 30s.

[0036] Further, the extracted nucleic acid can be stored at 4℃ for 48h.

[0037] The application of the nucleic acid extraction solution in the extraction of nucleic acid from plant leaves, the plant types of plant leaves include corn, tomato, blackberry and artichoke.

[0038] The nucleic acid extraction liquid is used for plant leaf nucleic acid extraction, and then ultrapure water is added, fully shaken and mixed, centrifuged to obtain supernatant, and the supernatant is added with pure water for genotyping detection.

[0039] Further, the centrifugal speed is 3500-3600 rmp, and the time is 4-5 min.

[0040] Compared with the prior art, the nucleic acid extraction liquid has the following advantages and beneficial effects:

[0041] 1. The nucleic acid extraction liquid directly combines specific concentrations of Tris-HCl, EDTA and SDS to extract plant leaf nucleic acid, has the advantages of simple formula, and can obtain sufficient DNA for subsequent PCR experiments using the simple extraction liquid, and the components of the extraction liquid are simpler without reducing the extraction effect compared with the existing extraction method.

[0042] 2. The nucleic acid extraction liquid has the advantages of simple work flow and short time for plant leaf nucleic acid extraction, uses a 96-well plate, and the extraction time of a single plate is only 15 minutes, and the extracted nucleic acid does not need to be detected for concentration and purity.

[0043] 3. The nucleic acid extraction liquid has the advantage of high detection accuracy for plant genotyping PCR detection: the site detection rate is ≥98%, and the genotype detection accuracy is ≥99.5%.

[0044] 4. The nucleic acid extraction liquid has high adaptability to different types of samples: compatible with a variety of plant leaf sample processing, suitable for low initial sample input, and stable performance. DETAILED DESCRIPTION

[0045] The accompanying drawings described herein are used to provide further understanding of the embodiments of the present application, constitute a part of the present application, and do not constitute a limitation of the embodiments of the present application. In the drawings:

[0046] Figure 1 The flow chart for plant leaf nucleic acid extraction of the present application;

[0047] Figure 2 is a fluorescence PCR detection diagram of a corn leaf sample extracted by the nucleic acid extraction liquid in the present application;

[0048] Figure 3 is a fluorescence PCR detection diagram of a fresh corn leaf sample extracted by the nucleic acid extraction liquid in the present application;

[0049] Figure 4 is a fluorescence PCR detection diagram of a tissue culture seedling leaf sample extracted by the nucleic acid extraction liquid in the present application;

[0050] Figure 5 These are fluorescence PCR detection images of corn leaf samples with different numbers of leaves extracted using the nucleic acid extraction solution of this invention. Among them, A is a fluorescence PCR detection image of a single corn leaf sample; B is a fluorescence PCR detection image of a four-corn leaf sample.

[0051] Figure 6 A shows a fluorescence PCR detection image of a corn leaf sample extracted with the nucleic acid extraction solution of this invention and stored at 4℃ for 0 h; B shows a fluorescence PCR detection image of a corn leaf sample extracted with the nucleic acid extraction solution of this invention and stored at 4℃ for 24 h; C shows a fluorescence PCR detection image of a corn leaf sample extracted with the nucleic acid extraction solution of this invention and stored at 4℃ for 48 h.

[0052] Figure 7 This is a fluorescence PCR detection image of a tomato sample extracted using the nucleic acid extraction solution in this invention;

[0053] Figure 8 This is a fluorescence PCR detection image of a blackberry sample extracted using the nucleic acid extraction solution in this invention.

[0054] Figure 9 This is a fluorescence PCR detection image of an artichoke sample extracted with nucleic acid extraction solution used in this invention.

[0055] Figure 10 This is a fluorescence PCR detection image of the corn sample in Comparative Example 1;

[0056] Figure 11 This is a fluorescence PCR detection image of the corn sample in Comparative Example 2;

[0057] Figure 12 This is a fluorescence PCR detection image of SDS concentration 0.3% in Example 9;

[0058] Figure 13 This is a fluorescence PCR detection image of an SDS concentration of 0.5% in Example 9;

[0059] Figure 14 This is a fluorescence PCR detection image of an SDS concentration of 0.6% in Example 9;

[0060] Figure 15 This is a fluorescence PCR detection image of SDS concentration 0.8% in Example 9;

[0061] Figure 16 This is a fluorescence PCR detection image of EDTA at a concentration of 10 mM in Example 10;

[0062] Figure 17 This is a fluorescence PCR detection image of EDTA at a concentration of 20 mM in Example 10;

[0063] Figure 18 is a fluorescence PCR detection graph of Example 10 in which the EDTA concentration is 30 mM;

[0064] Figure 19 is a fluorescence PCR detection graph of Example 11 in which the Tris-HCl concentration is 50 mM;

[0065] Figure 20 is a fluorescence PCR detection graph of Example 11 in which the Tris-HCl concentration is 60 mM;

[0066] Figure 21 is a fluorescence PCR detection graph of Example 11 in which the Tris-HCl concentration is 70 mM;

[0067] Figure 22 is a result graph of KASP detection procedure 1 of Example 12;

[0068] Figure 23 is a result graph of KASP detection procedure 2 of Example 12;

[0069] Figure 24 is a result graph of KASP detection procedure 3 of Example 12;

[0070] Figure 25 is a result graph of Taqman detection procedure 1 of Example 12;

[0071] Figure 26 is a result graph of Taqman detection procedure 2 of Example 12. DETAILED DESCRIPTION

[0072] In order to make the objects, technical solutions, and advantages of the present application clearer, further detailed description will be given to the present application combined with embodiments and drawings. The schematic embodiments of the present application and the description thereof are only used to explain the present application, and do not limit the present application. The embodiments described below are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0073] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one of ordinary skill in the art that the present application can be practiced without these specific details. In other embodiments, in order to avoid obscuring the present application, well-known structures, materials, or methods are not specifically described. The materials, instruments, and reagents used in the following embodiments, and the like, can be obtained from commercial channels unless otherwise specified. The technical means used in the embodiments, and the like, are conventional means known to those of ordinary skill in the art unless otherwise specified.

[0074] In addition, the terms "first", "second", etc. are used only for descriptive purposes and are not to be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can include one or more of the features explicitly or implicitly. In the description of the present application, the meaning of "a plurality" is two or more, unless otherwise explicitly and specifically limited.

[0075] Embodiment:

[0076] The present embodiment is based on providing a non-toxic, harmless, simple operation, low cost and easy to operate nucleic acid extraction method, and a nucleic acid extraction solution suitable for plant leaves is designed, which is prepared from 60 mM Tris-HCl, 20 mM EDTA and 0.5% SDS (mass / volume ratio). The nucleic acid extraction solution has the advantages of simple formula, and sufficient DNA can be obtained for subsequent PCR experiment by using simple extraction solution. Compared with the existing extraction method, the composition of the extraction solution is simpler without reducing the extraction effect.

[0077] Based on the above-mentioned nucleic acid extraction solution, the rapid extraction method of plant leaf nucleic acid is as shown in Figure 1 The method comprises the following steps:

[0078] 1) Using a punch to sample 1-4 pieces (0.4-1.6 mg), placing the obtained sample in a 96-well deep well plate or a field sampling tube, using a 5mm diameter steel ball to break, and obtaining a powder;

[0079] 2) Add 100 μL nucleic acid extraction solution to the powder after pretreatment, vortex, mix well after vortexing, 90℃ water bath for 10 min, and shake for 30 s every 5 min, then the released nucleic acid can be obtained.

[0080] The nucleic acid obtained by the present embodiment can be applied to subsequent fluorescent PCR genotyping method, which is realized by the following steps: adding 100 μL ultrapure water to the nucleic acid extraction solution, fully shaking and mixing, centrifuging at 3500-3600 rpm for 4-5 min, and then adding 5 μL supernatant to 95 μL ultrapure water for genotyping detection.

[0081] The nucleic acid obtained by the present embodiment can be stored at 4℃ for 48h, and can be tested multiple times during PCR genotyping.

[0082] The plant types of the plant leaves of the present embodiment include corn, tomato, blackberry and artichoke.

[0083] Because the concentrations of sugars, fats and some ions in seeds, roots and stems are different, the present method cannot be used for rapid extraction of other tissue types, and can only be used for plant leaves.

[0084] The embodiment can reduce the use of toxic reagents in the nucleic acid extraction process, simplify the nucleic acid extraction steps, greatly shorten the nucleic acid extraction and purification time, and significantly improve the detection throughput.

[0085] In order to better illustrate the technical effect of the embodiment, the following specific cases are used for illustration:

[0086] Embodiment 1:

[0087] The above nucleic acid extraction solution is used for corn dry leaf detection, including the following steps:

[0088] 1) Use a punch to take 1 piece of corn dry leaf;

[0089] 2) After crushing the leaf, add 100 microliters of nucleic acid extraction solution, and heat in a 90°C water bath for 10 minutes;

[0090] 3) After adding 100 microliters of ultrapure water, mix thoroughly, and take 5 microliters of supernatant into 95 microliters of ultrapure water to obtain diluted genomic DNA;

[0091] 4) Use the diluted DNA for single-point typing detection (fluorescent PCR typing method).

[0092] The fluorescent PCR detection results of single-point typing detection in this embodiment are shown in Table 1: Figure 2 After DNA sequencing verification, the genotyping detection rate of 200 samples was 100%, and the typing accuracy was 100%.

[0093] Embodiment 2:

[0094] The above nucleic acid extraction solution is used for corn fresh leaf detection, including the following steps:

[0095] 1) Use a punch to take 1 piece of corn fresh leaf;

[0096] 2)-4) The steps are the same as in Embodiment 1.

[0097] The fluorescent PCR detection results of single-point typing detection in this embodiment are shown in Table 2: Figure 3 After DNA sequencing verification, the genotyping detection rate of 200 samples was 100%, and the typing accuracy was 100%.

[0098] Embodiment 3:

[0099] The above nucleic acid extraction solution is used for corn tissue culture seedling detection, including the following steps:

[0100] 1) Use a punch to take 1 piece of corn tissue culture seedling, about 0.4-1.6 mg;

[0101] 2)-4) The steps are the same as in Embodiment 1.

[0102] The results of the single-point typing detection by fluorescent PCR detection are shown in Table 2. Figure 4 As shown in Table 2, the detection rate of genotyping of 200 samples was 100%, and the accuracy of genotyping was 100% after DNA sequencing verification.

[0103] Example 4:

[0104] The detection of the sampling amount of corn leaves included the following steps:

[0105] 1) Take 1-4 pieces of corn leaves, about 0.4-1.6 mg, using a puncher;

[0106] 2)-4) The steps are the same as in Example 1.

[0107] The results of the single-point typing detection by fluorescent PCR detection are shown in Table 2. Figure 5 As shown in Table 2, the detection rate of genotyping of 200 samples was 100%, and the accuracy of genotyping was 100% after DNA sequencing verification.

[0108] Example 5:

[0109] The detection of the storage time of genomic DNA included the following steps:

[0110] 1) Take 1 piece of corn leaf using a puncher;

[0111] 2) After crushing the leaf, add 100 microliters of nucleic acid extraction solution, and incubate at 90°C for 10 min;

[0112] 3) After adding 100 microliters of water, mix thoroughly, and take 5 microliters of supernatant into 95 microliters of water to obtain diluted genomic DNA;

[0113] 4) Use the diluted DNA for single-point typing detection;

[0114] 5) Test again after storing the genomic DNA at 4°C for 24h and 48h.

[0115] The results are shown in Table 2. Figure 6 As shown in Table 2, the detection rate of genotyping of 200 samples was 100%, and the accuracy of genotyping was 100% after DNA sequencing verification.

[0116] Example 6:

[0117] The detection of tomato leaves included the following steps:

[0118] 1) Take 1 piece of fresh tomato leaf using a puncher;

[0119] 2)-4) The steps are the same as in Example 1.

[0120] The results of the single-point typing detection by fluorescent PCR in this example are shown in Table 1. Figure 7 As shown in Table 1, after DNA sequencing verification, the genotyping detection rate of 83 samples was 100%, and the typing accuracy was 100%.

[0121] Example 7

[0122] The blackberry leaf detection included the following steps:

[0123] 1) Take one fresh blackberry leaf using a puncher;

[0124] 2)-4) The steps are the same as in Example 1.

[0125] The results of the single-point typing detection by fluorescent PCR in this example are shown in Table 1. Figure 8 As shown in Table 1, after DNA sequencing verification, the genotyping detection rate of 94 samples was 99%, and the typing accuracy was 100%.

[0126] Example 8

[0127] The artichoke leaf detection included the following steps:

[0128] 1) Take one fresh artichoke leaf using a puncher;

[0129] 2)-4) The steps are the same as in Example 1.

[0130] The results of the single-point typing detection by fluorescent PCR in this example are shown in Table 1. Figure 9 As shown in Table 1, after DNA sequencing verification, the genotyping detection rate of 285 samples was 100%, and the typing accuracy was 100%.

[0131] The above examples used the following fluorescent PCR typing method:

[0132] 1) PCR was performed using PACE 2.0 and primers designed for detecting the site;

[0133] 2) The PCR program is shown in Table 1:

[0134] Table 1

[0135]

[0136] 3) High-throughput genotyping system was used for PCR and detection of fluorescence values.

[0137] To confirm the extraction scheme, we performed the following test verification experiments:

[0138] In general, compared with Example 1, two additional different extraction schemes were set up simultaneously, namely Comparative Example 1 and Comparative Example 2. The main test scheme and test results are as follows (the samples with yellow circles in the typing chart are DNA control samples):

[0139] Comparative Example 1:

[0140] The nucleic acid extraction solution is formulated as follows:

[0141] A solution: 200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5% SDS;

[0142] B solution: 0.4 M guanidine hydrochloride.

[0143] Extraction procedure:

[0144] (1) Take one piece of corn leaf, grind for 1 min to break it up, add 100 ul (80 uL solution A + 20 uL solution B) of extraction solution, mix thoroughly for 30 s, and centrifuge at 12000 rpm for 1 min.

[0145] (2) Dilute 10 ul of supernatant with 50 times of enzyme-free sterile water.

[0146] (3) Use the diluted nucleic acid product for single-point typing experiments. The specific operation is as follows:

[0147] ① Use PACE2.0 and primers designed to detect the site for PCR;

[0148] ② The PCR program is shown in Table 1;

[0149] ③ Use high-throughput genotyping system for PCR and detection of fluorescence value.

[0150] The detection results are shown in Table 2. Figure 10 Comparative Example 2:

[0151] The nucleic acid extraction solution is formulated as follows:

[0152] A solution: 150 mM NaCl, 100 mM Sucrose, 25 mM EDTA, 200 m M Tris-HCl PH 7.5, 0.5% SDS;

[0153] B solution: 0.4 M guanidine hydrochloride.

[0154] Extraction procedure:

[0155] (1) Take one piece of corn leaf, grind for 1 min to break it up, add 100 ul (80 uL solution A + 20 uL solution B) of extraction solution, mix thoroughly for 30 s, and centrifuge at 12000 rpm for 1 min.

[0156] ​

[0157] (2) Take 10 ul supernatant and dilute 50 times with enzyme-free sterile water;

[0158] (3) Use the diluted nucleic acid product for single-point typing experiment. The specific single-point detection operation is the same as that of Comparative Example 1, and the results are shown in Table 3. Figure 11

[0159] From the results of different schemes, it can be concluded that, in addition to the DNA control sample, the nucleic acid obtained by the rapid extraction method in Comparative Examples 1 and 2 has poor typing effect, and the genotype data cannot be read according to the results. Figure 10 Figure 11

[0160] Example 9:

[0161] In order to verify the performance under different SDS concentrations in Example 1, the following experiments were performed:

[0162] 1) Use a punch to take one piece of dry corn leaf;

[0163] 2) After crushing the leaf, add 100 microliters of nucleic acid extraction solution, and incubate at 90°C for 10 minutes;

[0164] 3) After adding 100 microliters of ultrapure water, mix thoroughly, and take 5 microliters of supernatant into 95 microliters of ultrapure water to obtain diluted genomic DNA;

[0165] 4) Use the diluted DNA for single-point typing detection (fluorescent PCR typing method).

[0166] In this example, the nucleic acid extraction solution is prepared from 60 mM Tris-HCl, 20 mM EDTA, and 0.3%, 0.5%, 0.6%, or 0.8% SDS by mass / volume.

[0167] The fluorescent PCR detection results of single-point typing detection of 0.3%, 0.5%, 0.6%, or 0.8% SDS are shown in Table 4. Figures 12-15

[0168] From the results of different schemes, it can be concluded that, in addition to the DNA control sample, the nucleic acid obtained by the rapid extraction method in Comparative Examples 1 and 2 has poor typing effect, and the genotype data cannot be read according to the results. Figures 12-15

[0169] When the concentrations of the remaining components remain unchanged, the concentration of SDS is 0.5%, and the nucleic acid obtained using the nucleic acid extraction solution has the best typing effect in single-point typing detection, which is consistent with the typing effect of the control DNA. In the test results, when the concentration of SDS is 0.5%, the detection rate and the accuracy of genotype typing are both 100%.

[0170] Example 10:

[0171] ​​​​​In order to verify the performance under different concentrations of EDTA, the following experiments were carried out:

[0172] 1) Take one piece of dry corn leaf with a puncher;

[0173] 2) After crushing the leaf, add 100 microliters of nucleic acid extraction solution, and water bath at 90°C for 10 minutes;

[0174] 3) After adding 100 microliters of ultrapure water, mix thoroughly, and take 5 microliters of supernatant into 95 microliters of ultrapure water to obtain diluted genomic DNA;

[0175] 4) Use the diluted DNA for single-point typing detection (fluorescent PCR typing method).

[0176] In this embodiment, the nucleic acid extraction solution is prepared from 60 mM Tris-HCl, 0.5% SDS (mass / volume ratio), and 10 mM, 20 mM or 30 mM EDTA.

[0177] The results of single-point typing detection by fluorescent PCR detection of 10 mM, 20 mM and 30 mM EDTA are shown in Figures 16-18 .

[0178] It can be seen from Figures 16-18 that:

[0179] When the concentrations of the remaining components remain unchanged, the concentration of EDTA is 20 mM, the nucleic acid obtained using the nucleic acid extraction solution has the best typing effect in single-point typing detection, which is consistent with the typing effect of the control DNA. In the test results, when the concentration of EDTA is 20 mM, the detection rate and genotype typing accuracy are both 100%.

[0180] Example 11:

[0181] In order to verify the performance under different concentrations of Tris-HCl, the following experiments were carried out:

[0182] 1) Take one piece of dry corn leaf with a puncher;

[0183] 2) After crushing the leaf, add 100 microliters of nucleic acid extraction solution, and water bath at 90°C for 10 minutes;

[0184] 3) After adding 100 microliters of ultrapure water, mix thoroughly, and take 5 microliters of supernatant into 95 microliters of ultrapure water to obtain diluted genomic DNA;

[0185] 4) Use the diluted DNA for single-point typing detection (fluorescent PCR typing method).

[0186] In the embodiment, the nucleic acid extraction solution is prepared from 50 mM, 60 mM or 70 mM Tris-HCl, 20 mM EDTA and 0.5% SDS (mass / volume ratio).

[0187] The results of the single-point genotyping detection of 50 mM, 60 mM and 70 mM Tris-HCl are shown in FIGS. 2, 3 and 4, respectively. Figures 19-21

[0188] It can be seen from FIGS. 2, 3 and 4 that: Figures 19-21

[0189] When the concentrations of the other components remain unchanged, the nucleic acid obtained by using the nucleic acid extraction solution has the best genotyping effect in the single-point genotyping detection when the concentration of Tris-HCl is 60 mM, which is consistent with the genotyping effect of the control DNA. In the test results, the detection rate and the genotyping accuracy are both 100% when the concentration of Tris-HCl is 60 mM.

[0190] It can be seen from the results of different gradients that the lysis solution prepared from 60 mM Tris-HCl, 20 mM EDTA and 0.5% SDS (mass / volume ratio) has the best extraction effect.

[0191] Embodiment 12

[0192] Test of different amplification procedures suitable for the extraction scheme in this embodiment.

[0193] The specific test process is as follows

[0194] Using 96 samples, sample into a 96-well plate, use a puncher to take 1 piece of dry corn leaf, crush the leaf, add 100 microliters of nucleic acid extraction solution, 90°C water bath for 10 min; after adding 100 microliters of ultrapure water, shake well to mix, take 5 microliters of supernatant into 95 microliters of ultrapure water to obtain diluted genomic DNA; use the diluted DNA for single-point genotyping detection (fluorescent PCR genotyping method), and the single-point genotyping test procedure for crude extraction is as follows:

[0195] Different KASP detection procedures are shown in Table 2:

[0196] Table 2

[0197]

[0198] The test results of KASP detection procedures 1-3 are shown in FIGS. 5, 6 and 7, respectively. Figures 22-24

[0199] Different Taqman detection procedures are shown in Table 3 and Table 4, respectively:

[0200] Table 3

[0201] ​​​

[0202] Table 4

[0203]

[0204] The test results of Taqman testing programs 1-2 are as follows: Figures 25-26 As shown.

[0205] Depend on Figures 22-26 It can be known that:

[0206] During the program testing, KASP detection program 2, KASP detection program 3, and Taqman detection program 3 all had poor genotyping and some samples did not amplify. KASP detection program 1 and Taqman detection program 1 had clear genotyping and the detection rate and genotyping accuracy were both 100%.

[0207] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nucleic acid extraction solution suitable for use on plant leaves, characterised in that, Prepared from 60 mM Tris-HCl, 20 mM EDTA and 0.5% SDS by mass / volume.

2. A method for rapid extraction of nucleic acids from plant leaves based on the nucleic acid extraction solution according to claim 1, characterized by, The method comprises the following steps: After the plant leaves are broken, the broken pieces are obtained, a nucleic acid extraction solution is added to the broken pieces, and the mixture is shaken and mixed and subjected to high-temperature water bath, so that the extracted nucleic acid is obtained.

3. The method of flash extraction according to claim 2, wherein, The sample types of the plant leaves include fresh, dried plant seedling leaves and leaves obtained through tissue culture.

4. The method of flash extraction according to claim 3, wherein, The process of obtaining the broken pieces is as follows: 0.4-1.6 mg is sampled by using a puncher, the obtained sample is placed in a 96-well deep well plate or a field sampling tube, and a 5-mm-diameter steel ball is used to break the sample.

5. The method of flash extraction of claim 2, wherein, The temperature of the high-temperature water bath is 90°C, and the time is 10 min.

6. The method of rapid extraction of claim 5, wherein, During the high-temperature water bath process, the mixture is shaken and mixed for 30 s every 5 min.

7. The method of flash extraction of claim 2, wherein, The obtained nucleic acid can be stored at 4°C for 48 h.

8. Use of the nucleic acid extraction solution according to claim 1 in the extraction of nucleic acids from plant leaves, characterized in that, The plant types of the plant leaves include corn, tomato, blackberry and artichoke.

9. Use of the nucleic acid extraction solution according to claim 1 in genotyping PCR detection, characterized in that, The nucleic acid of the plant leaves is extracted by using the nucleic acid extraction solution, then ultrapure water is added, the mixture is shaken and mixed sufficiently, centrifuged to obtain supernatant, and pure water is added for genotyping detection.

10. Use according to claim 9, characterized in that, The centrifugation speed is 3500-3600 rmp, and the time is 4-5 min.

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