Fungus nucleic acid lysate, nucleic acid extraction kit and extraction method

Abstract

The invention discloses a fungal nucleic acid lysate, a nucleic acid extraction kit and an extraction method, the fungal nucleic acid lysate can be combined with lyase to fully dissolve and break cell walls of fungi, so that nucleic acid is fully released to realize combination with magnetic beads, and the concentration and purity of nucleic acid extraction are greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid extraction, in particular to a fungal nucleic acid lysate, a nucleic acid extraction kit and an extraction method. Background Art

[0002] Currently, invasive fungal diseases (IFDs) remain a key cause of high morbidity and mortality in many immunosuppressed patients. According to statistics, the total number of people who die from fungal infections each year has risen to 3.75 million worldwide, twice the previous estimate. Accurate etiology diagnosis and effective treatment are extremely critical for severe or critical IFDs. However, accurate diagnosis of IFDs still faces many difficulties.

[0003] Commonly used diagnostic methods for fungal infections include direct microscopy, fungal culture, serological testing, and molecular biological testing. Direct microscopy uses a microscope to observe whether there are fungal hyphae or spores in the sample. This method is quick and simple, but the sensitivity is poor; the fungal culture method is regarded as the gold standard for pathogen diagnosis, but its culture cycle is long and it is difficult to cultivate some fungi. Serological testing methods are rich and diverse, and can detect fungal-specific antibodies (such as Candida antibodies, Aspergillus antibodies, etc.) and fungal antigens (such as Candida mannan antigens, Aspergillus galactomannan antigens, etc.), and occupy an important position in the diagnosis of fungal infections. However, this method also has a certain proportion of false positive and false negative results, and is limited by the principle of detection technology. It can only identify the fungal genus level and cannot further distinguish the differences between fungal species. Molecular biological detection techniques, such as polymerase chain reaction (PCR) and related technologies, have high sensitivity and specificity, and can quickly detect fungal DNA or RNA, but cannot achieve high-throughput detection.

[0004] With the development of molecular technology in recent years, high-throughput sequencing technology has been increasingly used in IFDs. High-throughput sequencing can directly extract nucleic acid information from various specimens without the need for culture and hypothesis. It can detect all potential pathogens, especially for identifying unknown, rare, emerging or mixed infections.

[0005] Given the complexity of clinical samples, the diversity of pathogens, and the many forms of existence, the efficient extraction of fungal pathogen nucleic acids faces many challenges, especially in samples from patients with invasive fungal infections, where it is extremely difficult to break the fungal cell wall, which further increases the difficulty of efficiently extracting fungal pathogen nucleic acids. Whether it is molecular diagnosis or high-throughput sequencing, the premise is to extract sufficient and high-quality nucleic acids. To obtain sufficient and high-quality nucleic acids, the fungal cell walls must be fully broken so that the nucleic acids are fully released and bound to the magnetic beads. Due to the characteristics of the fungal cell wall, the existing lysis methods still have many shortcomings. Summary of the invention

[0006] The present invention provides a fungal nucleic acid lysing solution, a nucleic acid extraction kit and an extraction method. The fungal nucleic acid lysing solution can fully dissolve and break the cell wall of the fungus, so that the nucleic acid is fully released to achieve combination with magnetic beads, thereby improving the quality of nucleic acid extraction.

[0007] In view of this, the scheme of the present invention is:

[0008] The first aspect of the present invention is to provide a fungal nucleic acid lysing solution, which is composed of Tris-HCl with a molar concentration of 0.004-0.005 mol / L, sodium hydroxide with a molar concentration of 0.003-0.004 mol / L, and polyethylene glycol 200 with a volume percentage of 55%-60%, sodium N-lauroyl sarcosinate with a volume percentage of 1.0-1.2%, sodium perchlorate with a volume percentage of 1.8-2%, and sorbitol fatty acid ester with a volume percentage of 1.8-2%.

[0009] Furthermore, the fungal nucleic acid lysate consists of 0.0045 mol / L Tris-HCl, 0.0035 mol / L sodium hydroxide, 60% by volume of polyethylene glycol 200, 1.0% of sodium N-lauroyl sarcosinate, 2% of sodium perchlorate and 2% of sorbitan fatty acid ester.

[0010] Furthermore, the pH of the fungal nucleic acid lysate is 6.1-6.3.

[0011] The second aspect of the present invention is to provide a fungal nucleic acid extraction kit, comprising the fungal nucleic acid lysing solution described in the first aspect, as well as a lysing enzyme, a magnetic bead solution, a washing solution and an elution solution.

[0012] Furthermore, in the above-mentioned fungal nucleic acid extraction kit:

[0013] The lyase is Zymolyase;

[0014] The magnetic bead liquid comprises magnetic beads and water;

[0015] The washing solution includes 0.003 mol / L Tris-HCl, 0.02 mol / L NaCl, and 80% by volume of anhydrous ethanol;

[0016] The eluent includes 0.01 mol / L Tris-HCl.

[0017] Further, the pH value of the washing solution is 7.1-7.3; and / or the pH value of the eluent is adjusted to 8.5-8.7.

[0018] The third aspect of the present invention is to propose the use of the fungal nucleic acid lysis solution described in the first aspect or the fungal nucleic acid extraction kit described in the second aspect in fungal nucleic acid extraction.

[0019] The fourth aspect of the present invention is to provide a method for extracting fungal nucleic acid, using the fungal nucleic acid extraction kit described in the second aspect, the steps comprising:

[0020] S1. Mix the lytic enzyme, lysis solution and sample to break the cell wall and release nucleic acid, and then centrifuge at high speed to remove the precipitate and keep the supernatant;

[0021] S2. Add magnetic beads to the supernatant to adsorb nucleic acids;

[0022] S3. Wash the magnetic beads bound to the nucleic acid using a washing solution;

[0023] S4. Use an elution solution to elute the nucleic acid on the washed magnetic beads to obtain a fungal nucleic acid extract.

[0024] Furthermore, in step S1, the volume ratio of the lysis solution to the fungal liquid sample is (2-4):1; the temperature of the cell wall breaking and lysis process to release nucleic acid is 60-70°C and the time is 10-20min.

[0025] Furthermore, in step S1, the rotation speed of the high-speed centrifugal deprecipitation process is 12000-15000g, the time is 3-5min, and the temperature is 4-8°C.

[0026] Furthermore, in step S2, the nucleic acid adsorption process is performed at room temperature for 10-20 minutes.

[0027] Furthermore, in step S3, the washing times are 1-3 times, and the time required for each washing is 1-3 minutes.

[0028] Furthermore, in step S4, the elution process temperature is room temperature and the elution time is 1-3 minutes.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The nucleic acid lysing solution provided by the present invention can be combined with a lysing enzyme to fully dissolve and break the cell wall of the fungus, so that the nucleic acid is fully released to achieve binding with magnetic beads, thereby greatly improving the concentration and purity of nucleic acid extraction.

[0031] The lysing enzyme and lysing solution used in the fungal nucleic acid extraction kit provided by the present invention can fully dissolve and break the cell wall of the fungus, so that the nucleic acid is fully released and combined with the magnetic beads, which significantly improves the efficiency and quality of nucleic acid extraction. In addition, the kit has simple components and raw materials, simple operation, and no toxic reagents are used, which is healthy and safe. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only 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.

[0033] In one embodiment, a fungal nucleic acid extraction kit is provided, comprising a lysing enzyme, a lysing solution, a magnetic bead solution, a washing solution, and an elution solution; wherein:

[0034] The lyase is Zymolyase;

[0035] The lysing solution comprises: the lysing solution comprises: 0.004-0.005 mol / L Tris-HCl, 0.003-0.004 mol / L sodium hydroxide, and 55-60% by volume of polyethylene glycol 200, 1.0-1.2% of sodium N-lauroyl sarcosinate, 1.8-2% of sodium perchlorate and 1.8-2% of sorbitol fatty acid ester;

[0036] The magnetic bead liquid comprises: magnetic beads and water;

[0037] The washing solution comprises: 0.0028-0.0032 mol / L Tris-HCl, 0.018-0.022 mol / L NaCl, and 70-80% by volume of anhydrous ethanol;

[0038] The eluent includes: 0.008-0.01 mol / L Tris-HCl.

[0039] In the above embodiment, the nucleic acid lysis solution can combine with the lysis enzyme to fully dissolve and break the cell wall of the fungus, so that the nucleic acid is fully released to achieve binding with the magnetic beads, thereby greatly improving the concentration and purity of the nucleic acid extraction.

[0040] In a preferred embodiment, for the components in the lysate, the concentration of Tris-HCl may be, for example, 0.0041, 0.0042, 0.0043, 0.0044, 0.0045, 0.0046, 0.0047, 0.0048, 0.0049 or 0.005 mol / L; the concentration of 0.003-0.004 mol / L sodium hydroxide may be, for example, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039 or 0.004 mol / L; 55-60% by volume of polyethylene glycol 200 may be, for example, 55, 56, 57, 58, 59 or 60%; 1.0-1.2% sodium N-lauroyl sarcosinate may be, for example, 1.0, 1.1 or 1.2; the volume ratio of sodium perchlorate may be, for example, 1.8, 1.9 or 2.0; and sorbitan fatty acid ester may be, for example, 1.8, 1.9 or 2.0.

[0041] In a preferred embodiment, in the washing solution, the concentration of Tris-HCl can be, for example, 0.0028, 0.0029, 0.003, 0.0031 or 0.0032 mol / L; the concentration of NaCl can be, for example, 0.018, 0.019, 0.02, 0.021 or 0.022 mol / L; the concentration of anhydrous ethanol can be, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%.

[0042] In a preferred embodiment, the lysate includes 0.0045 mol / L Tris-HCl, 0.0035 mol / L sodium hydroxide, and 60% by volume of polyethylene glycol 200, 1.0% sodium N-lauroyl sarcosinate, 2% sodium perchlorate and 2% sorbitan fatty acid ester; the pH of the lysate is 6.1-6.3; the pH of the washing solution is 7.1-7.3; and the pH of the eluent is 8.5-8.7.

[0043] In another embodiment, a method for extracting fungal nucleic acid is provided, wherein the fungal nucleic acid extraction kit is used for extraction, and the steps include:

[0044] S1: Mix the lysing enzyme, lysing solution and sample to break the fungus wall and release nucleic acid, remove the precipitate by high-speed centrifugation and keep the supernatant; the volume ratio of lysing solution to fungal culture liquid can be (2-4):1;

[0045] S2: Add magnetic bead solution to the supernatant and keep it at room temperature for 10-20 minutes to allow the nucleic acid to fully bind to the magnetic beads;

[0046] S3: Use washing solution to wash the magnetic beads bound to nucleic acid; the number of washes is 1 to 3 times, and each wash time can be selected to be 1-3 minutes;

[0047] S4: Using an elution solution to elute the nucleic acid on the washed magnetic beads to obtain a fungal nucleic acid extract.

[0048] In a preferred embodiment, the time required for lysis in step S1 is 10-20 min, for example, it can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min; the temperature required for lysis is 60-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C.

[0049] In a preferred embodiment, in step S2, the supernatant is retained after the precipitate is removed by high-speed centrifugation, the required time is 3-5 minutes, the temperature is 4-8°C, and the rotation speed is 12000g-15000g, such as 12000g, 13000g, 14000g or 15000g.

[0050] In a preferred embodiment, various raw materials are accurately weighed / measured to prepare the eluent according to the eluent ratio of the above-mentioned fungal nucleic acid extraction kit. The time required for eluting the nucleic acid on the washed magnetic beads in this step is 1 to 3 minutes, for example, 1 minute, 2 minutes or 3 minutes; the temperature is 50 to 60°C, for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C.

[0051] The following specific implementation examples are used to illustrate the technical effects of the above nucleic acid lysing solution or nucleic acid extraction kit. Unless otherwise specified, the reagents involved are common commercially available standards in the field, and the methods involved are experimental means that are well-known to those skilled in the art.

[0052] Example 1

[0053] The lysis solution 1 is composed of: 0.0045 mol / L Tris-HCl, 0.0035 mol / L sodium hydroxide, and 60% by volume of polyethylene glycol 200, 1.0% of sodium N-lauroyl sarcosinate, 2% of sodium perchlorate, and 2% of sorbitan fatty acid ester.

[0054] Example 2

[0055] Lysis solution 2 is composed of: 0.004 mol / L Tris-HCl, 0.003 mol / L sodium hydroxide, and 55% by volume of polyethylene glycol 200, 1.0% sodium N-lauroyl sarcosinate, 1.8% sodium perchlorate, and 1.8% sorbitan fatty acid ester.

[0056] Example 3

[0057] Lysis solution 3 is composed of: 0.005 mol / L Tris-HCl, 0.004 mol / L sodium hydroxide, and 58% by volume of polyethylene glycol 200, 1.2% of sodium N-lauroyl sarcosinate, 1.9% of sodium perchlorate, and 1.9% of sorbitan fatty acid ester.

[0058] Experimental Example 1 Nucleic Acid Extraction

[0059] 1. Extraction Method

[0060] Other related reagents include: Zymolyase is a lytic enzyme; the magnetic bead solution is magnetic beads and water; the washing solution includes 0.003 mol / L Tris-HCl, 0.02 mol / L NaCl, and 80% by volume of anhydrous ethanol; and the eluting solution is 0.01 mol / L Tris-HCl.

[0061] 1) Take 200 μL of fungal culture sample, add 10 μL of lysing enzyme and 400 μL of lysing solution, shake and mix, incubate at 60°C for 10 min, and centrifuge at 15000 g for 2 min.

[0062] 2) Take 500 μL of the supernatant after centrifugation in S1 and add it to a new PE tube and add 20 μL of magnetic bead solution. After oscillation and mixing, let it stand at room temperature for 10 minutes, place it on the magnetic stand for 5 minutes, and discard the supernatant.

[0063] 3) Add 500 μL of washing solution to resuspend the magnetic bead nucleic acid mixture, wash twice, and absorb the remaining liquid.

[0064] 4) Open the lid and let it stand at room temperature for 10 minutes, add 50 μL DNA elution buffer, shake and mix, and let it stand at room temperature for 10 minutes. Place it on the magnetic rack for 5 minutes, and transfer the supernatant to a new EP tube.

[0065] 2. Using the nucleic acid lysis solution provided in Examples 1-3, and using the same lytic enzyme, magnetic bead solution, washing solution and elution solution, nucleic acid extraction and nucleic acid performance test were performed on Candida albicans, Candida glabrata and Aspergillus terreus, respectively. Three samples were set for each group. Different lysis solutions were used to extract each group of biological samples while other components remained unchanged.

[0066] 3. Nucleic acid extraction quality test

[0067] 1) The nucleic acid concentration of the extracted biological samples is measured respectively. The specific detection method adopts a nucleic acid concentration detection kit that complies with GB / T40982-2021 "Quality Evaluation Requirements for Novel Coronavirus Nucleic Acid Detection Kits".

[0068] 2) The desired nucleic acid samples were used as starting templates and amplified by real-time fluorescence quantitative PCR (qPCR). Specific amplification primers (see Table 1) were used in the experiment, and amplification was performed under the optimized amplification reaction system (see Table 2) and reaction program (see Table 3). The number of cycles (Ct value) experienced when the fluorescence signal of the amplified product reached the set threshold was recorded and used for subsequent quantitative analysis.

[0069] Table 1: Amplification reaction primer system

[0070]

[0071] Table 2: Amplification reaction mixture system

[0072] Components Dosage SYBR Green qPCR Mix 12.5ul Upstream primer 0.5ul Downstream primer 0.5ul <![CDATA[Nuclease-Free ddH2O]]> 9.5ul template 2ul

[0073] Table 3: Amplification reaction program

[0074]

[0075] 4. Results of nucleic acid extraction quality test

[0076] The test results are shown in Table 5 below.

[0077] Table 5: Sample extraction nucleic acid concentration, purity test results, and fluorescence quantitative PCR amplification results

[0078]

[0079]

[0080] According to the test results in Table 5 above, it is found that the purity of nucleic acid extraction of the above samples is between 1.70-2.0, and the purity is good. The CT value of fluorescent quantitative PCR amplification is small, indicating that the nucleic acid extraction is successful, and the concentration and purity are both high.

[0081] Experimental Example 2 Effects of different components in lysate on nucleic acid extraction

[0082] This experimental example aims to determine the influence of different components in the lysate on the effect of nucleic acid extraction. According to the composition of the lysate 1 in Example 1, the components are adjusted to prepare a variety of different lysates. The components are shown in Table 6. The same biological sample (Candida albicans, Candida glabrata, and Aspergillus terreus bacterial samples are the same as those in Experimental Example 1) is extracted using the method of Experimental Example 1. After the extraction is completed, the concentration and purity of the obtained nucleic acid are determined using a nucleic acid concentration detection kit, and the Ct value is determined according to the fluorescent quantitative PCR method of Experimental Example 1. The experimental results are shown in Table 6.

[0083] Table 6: Sample nucleic acid concentration, purity and Ct value results

[0084]

[0085] It is not difficult to see from Table 6 that the concentration and purity of the nucleic acid extracted using lysis solution 1 are significantly better than those of the comparative lysis solution 1-2 and the nucleic acid extracted without using the lysis enzyme; the qPCR test results of the extracted sample DNA show that the extracted sample DNA has a CT value, indicating that the nucleic acid extraction is successful, but the CT value of the nucleic acid extracted using the comparative lysis solution 1-2 and without using the lysis enzyme is significantly greater than the CT value of the nucleic acid extracted using the lysis solution 1, indicating that the chemical composition and concentration of the lysis solution have a greater influence on the nucleic acid extraction effect and the lysis enzyme in the present invention has an influence on the nucleic acid extraction, and the Zymolyase lysis enzyme used can effectively dissolve the fungal cell wall and improve the quality of nucleic acid extraction.

[0086] Experimental Example 3 Effect of polyethylene glycol 200 concentration on nucleic acid extraction effect

[0087] This example aims to determine the effect of polyethylene glycol 200 concentration on nucleic acid extraction. A series of lysates were prepared by precisely controlling the concentration of polyethylene glycol 200. In each lysate, the concentration of polyethylene glycol 200 was 20%, 40%, 60%, 80%, and 100%, respectively, and the other components remained the same. The biological sample was treated with the nucleic acid extraction method described in Experimental Example 1. After the extraction was completed, the concentration and purity of the obtained nucleic acid were determined using a nucleic acid concentration detection kit, and the Ct value was determined according to the fluorescent quantitative PCR method of Example 1. The experimental results are shown in Table 7.

[0088] The results showed that the concentration of polyethylene glycol 200 in the lysis buffer had a significant effect on the concentration of extracted nucleic acids. When the concentration of polyethylene glycol 200 was 60%, the lysis effect was the best; when the concentration of PEG200 exceeded 60%, it was found that the solubility with other reagents was significantly reduced, affecting the subsequent experimental operation and the accuracy of the results.

[0089] Table 7: Sample nucleic acid concentration, purity and Ct value results

[0090]

[0091]

[0092] Experimental Example 4 Effect of Sodium N-Lauroyl Sarcosinate Concentration on Nucleic Acid Extraction

[0093] This example studies the effect of sodium N-lauroyl sarcosinate concentration on nucleic acid extraction. A series of lysates were prepared according to different sodium N-lauroyl sarcosinate concentrations, with concentrations of 0.5%, 1%, 5%, 10%, and 20%, respectively, and the remaining components remained unchanged. The biological sample was treated using the nucleic acid extraction method of Example 1, the concentration and purity of the obtained nucleic acid were determined using a nucleic acid concentration detection kit, and the Ct value was determined using the fluorescent quantitative PCR method of Example 1. The experimental results are shown in Table 8.

[0094] Table 8: Sample nucleic acid concentration, purity and Ct value results

[0095]

[0096] The results showed that the concentration of sodium N-lauroyl sarcosinate in the lysis solution had a significant effect on the concentration of extracted nucleic acids. When the concentration of sodium N-lauroyl sarcosinate was 1%, the lysis effect was good; when the concentration reached 10% or above, the concentration and purity of nucleic acids decreased significantly, affecting the quality of nucleic acids and subsequent experimental applications.

[0097] Experimental Example 5 Effect of Sodium Perchlorate Concentration on Nucleic Acid Extraction

[0098] This example discusses the effect of sodium perchlorate concentration on nucleic acid extraction. A series of lysates with sodium perchlorate concentrations of 0.5%, 2%, 5%, 10%, and 20% were prepared, and the other components were kept the same. The nucleic acid in the biological sample was extracted using the method of Example 1, and the concentration and purity of the obtained nucleic acid were determined using a nucleic acid concentration detection kit, and the Ct value was determined according to the fluorescent quantitative PCR method of Example 1. The experimental results are shown in Table 9.

[0099] Table 9: Results of nucleic acid concentration, purity and Ct value

[0100]

[0101] The results showed that the concentration of sodium perchlorate in the lysis solution had a significant effect on the concentration of extracted nucleic acids. When the sodium perchlorate concentration was 2%, the lysis effect was optimal; when the concentration reached 10% and above, the Ct value of nucleic acid amplification increased significantly, indicating that the efficiency of nucleic acid amplification decreased, affecting subsequent nucleic acid detection and analysis.

[0102] Experimental Example 6 Effect of Sorbitol Fatty Acid Ester Concentration on Nucleic Acid Extraction

[0103] This example explores the effect of sorbitol fatty acid ester concentration on nucleic acid extraction. Lysates with sorbitol fatty acid ester concentrations of 0.5%, 2%, 5%, 10%, and 20% were prepared, ensuring that the other components were the same. The biological sample nucleic acid was extracted according to the method of Example 1, the concentration and purity of the obtained nucleic acid were determined using a nucleic acid concentration detection kit, and the Ct value was determined according to the fluorescent quantitative PCR method of Example 1. The experimental results are shown in Table 10.

[0104] Table 10: Sample nucleic acid concentration, purity and Ct value results

[0105]

[0106] The results showed that the concentration of sorbitol fatty acid esters in the lysis solution had a significant effect on the concentration of nucleic acid extraction. When the concentration of sorbitol fatty acid esters was 2%, the lysis effect was the best; when the concentration reached 10% or above, the lysis solution showed precipitation that could not settle, interfering with the nucleic acid extraction process and affecting the nucleic acid quality.

[0107] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A fungal nucleic acid lysate, characterized in that: The fungal nucleic acid lysing solution consists of Tris-HCl with a molar concentration of 0.004-0.005 mol / L, sodium hydroxide with a molar concentration of 0.003-0.004 mol / L, polyethylene glycol 200 with a volume percentage of 55%-60%, sodium N-lauroyl sarcosinate with a volume percentage of 1.0-1.2%, sodium perchlorate with a volume percentage of 1.8-2% and sorbitol fatty acid ester with a volume percentage of 1.8-2%.

2. The fungal nucleic acid lysate according to claim 1, characterized in that The fungal nucleic acid lysing solution consists of Tris-HCl with a molar concentration of 0.0045 mol / L, sodium hydroxide with a molar concentration of 0.0035 mol / L, and polyethylene glycol 200 with a volume percentage of 60%, sodium N-lauroyl sarcosinate with a volume percentage of 1.0%, sodium perchlorate with a volume percentage of 2%, and sorbitol fatty acid ester with a volume percentage of 2%.

3. The fungal nucleic acid lysing solution according to claim 1 or 2, characterized in that: The pH of the fungal nucleic acid lysate is 6.1-6.

3.

4. A fungal nucleic acid extraction kit, characterized in that: The method comprises the fungal nucleic acid lysing solution according to claim 1, and a lysing enzyme, a magnetic bead solution, a washing solution and an elution solution.

5. The fungal nucleic acid extraction kit according to claim 4, characterized in that: The lyase is Zymolyase; And / or, the magnetic bead liquid comprises magnetic beads and water; And / or, the washing solution comprises 0.003 mol / L Tris-HCl, 0.02 mol / L NaCl, and 80% by volume of anhydrous ethanol; And / or, the eluent comprises 0.01 mol / L Tris-HCl.

6. The fungal nucleic acid extraction kit according to claim 4, characterized in that: The pH value of the washing solution is 7.1-7.3; and / or the pH value of the eluent is adjusted to 8.5-8.

7.

7. Use of the fungal nucleic acid lysate according to any one of claims 1 to 3 or the fungal nucleic acid extraction kit according to any one of claims 4 to 6 in extracting fungal nucleic acids.

8. A method for extracting fungal nucleic acid, using the fungal nucleic acid extraction kit according to any one of claims 4 to 6, characterized in that the steps include: S1. Mix the lytic enzyme, lysis solution and sample to break the cell wall and release nucleic acid, and then centrifuge at high speed to remove the precipitate and keep the supernatant; S2. Add magnetic beads to the supernatant to adsorb nucleic acids; S3. Wash the magnetic beads bound to the nucleic acid using a washing solution; S4. Use an elution solution to elute the nucleic acid on the washed magnetic beads to obtain a fungal nucleic acid extract.

9. The method for extracting fungal nucleic acid according to claim 8, characterized in that: In step S1: The sample is a fungal culture liquid, and the volume ratio of the lysate to the sample is (2-4):1; And / or, the temperature of the cell wall breaking and cleavage process to release nucleic acid is 60-70°C and the time is 10-20min; And / or, the rotation speed of the high-speed centrifugal deprecipitation process is 12000-15000g, the time is 3-5min, and the temperature is 4-8°C.

10. The method for extracting fungal nucleic acid according to claim 8, characterized in that: In step S2, the nucleic acid adsorption process is performed at room temperature for 10-20 minutes; And / or, in step S3, the washing times are 1-3 times, and the time required for each washing is 1-3 minutes; And / or, in step S4, the elution process temperature is room temperature and the elution time is 1-3 minutes.

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