Rapid and convenient method for large-scale extraction of fungus DNA

Abstract

The invention discloses a rapid and convenient method for extracting fungus DNA on a large scale, and belongs to the technical field of fungus DNA extraction.The method specifically comprises the steps that a set amount of fungus hyphae or sporocarps are taken and put into a 1.5 mL centrifugal tube; a set amount of Chelex-100 sodium form is added, and a set amount of Chelex-100 sodium form is added; adding 10-50L of sterile deionized water, and mashing the hyphae or sporocarp by using a grinding rod matched with a 1.5 mL centrifugal tube; adding 10 to 50 L of 10 * EX Taq buffer solution, and placing the centrifugal tube on a mixer for uniform mixing; boiling water bath; performing ice bath; performing centrifugal operation on the centrifugal tube after the ice bath; sucking supernate of the solution centrifuged by the centrifugal tube into a new centrifugal tube, and storing at-20 DEG C; according to the method, the number of extraction steps is small, time and labor are saved, extraction can be completed within 20 min under the condition that the extraction quality is guaranteed, extraction can be completed within 15 min at the soonest, and the effect is remarkable.

Description

Technical Field

[0001] The invention belongs to the technical field of fungal DNA extraction and relates to a fast and convenient method for extracting fungal DNA in large quantities. Background Art

[0002] Fungi, the oldest and most diverse group of organisms on Earth, are widely distributed in nature across various ecosystems and play a vital role. Some fungi, such as yeasts, are microscopic and can only be observed under a microscope; others, such as giant mushrooms and Ganoderma lucidum, are large and visible to the naked eye. Fungi are closely intertwined with human life. Edible fungi can provide a wealth of nutrients, meeting human health needs. The edible fungi industry also transforms and utilizes waste products such as crop straw. Natural biocontrol bacteria are highly effective in controlling plant diseases and insect pests. Metabolites of fungi, such as Penicillium, have therapeutic effects on human and animal diseases. Many pathogenic fungi can cause illness in humans and animals, and death in plants. Therefore, the correct classification of fungi is crucial for studying their physiological and ecological functions.

[0003] Fungi are numerous and diverse in nature, making identification using traditional classification methods challenging and often subject to errors. With the rapid development of molecular biology, accurate fungal classification and identification using these techniques has become essential. However, in molecular biology research, the extraction of fungal DNA is a fundamental prerequisite, and the rapid and efficient acquisition of fungal genomic DNA is crucial.

[0004] At present, many methods for DNA extraction have been reported, such as CTAB method, PTB method, kit method, SDS method, urea method, benzyl chloride method, etc. However, these methods are generally cumbersome and time-consuming (CTAB method takes 3 hours to complete, such as Figure 7 The extraction process shown in the figure) and high costs make the classification and identification of large numbers of fungal specimens prohibitively laborious, hindering rapid identification. Furthermore, PCR is sensitive to trace DNA concentrations, capable of amplifying even pg levels. Therefore, it is imperative to develop a rapid and convenient method for extracting genomic DNA from fungal fruiting bodies or hyphae to support rapid fungal classification and identification. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a fast and convenient method for extracting fungal DNA in large quantities, which has low extraction cost, high efficiency, and is very easy to operate. It can meet the needs of further research work such as PCR amplification and sequencing, and solve the problems commonly existing in conventional methods such as cumbersome operation process, long time consumption, and high cost.

[0006] The technical solution adopted by the present invention is a rapid and convenient method for extracting fungal DNA in large quantities, comprising the following steps: Step 1: Take a set amount of fungal hyphae or fruiting bodies and place them in a 1.5 mL centrifuge tube; Step 2. Add a predetermined amount of Chelex-100 sodium form (Coolaber, model: CC3631) to the centrifuge tube in step 1. Step 3: Add 10-50 µL of sterile deionized water to the centrifuge tube in step 2 and crush the mycelium or fruiting body with a grinding rod provided with a 1.5 mL centrifuge tube. Step 4. Add 10-50µL of 10×EX Taq buffer to the centrifuge tube in step 3 and mix thoroughly on a mixer. Step 5, place the centrifuge tube after mixing the solution in step 4 into a boiling water bath; Step 6: Place the centrifuge tube after the boiling water bath in step 5 in an ice bath; Step 7, centrifuging the centrifuge tube after ice bath in step 6; Step 8: Pipette the supernatant (containing DNA) from the solution after centrifugation in step 7 into a new centrifuge tube and store it at -20°C.

[0007] Furthermore, the mycelium or fruiting body in the above step 1 is selected from: green mold (Trichoderma sp.), big ball-cap mushroom (Strophariarugosoannulata), Ganoderma lucidum (Ganoderma lucidum), brown gray mushroom fruiting body (Tricholomaterreum), shiitake mushroom (Lentinula edodes), hairy wood ear (Auricularia polytricha), oyster mushroom (Pleurotus ostreatus), morel (Morchella esculenta (L.) Pers.), milk mushroom fruiting body (Lactarius sp.), gray tree flower (Grifola frondosa), sticky cover milk boletus (Suillus bovinus), pig mushroom (Panus gigianteus), velvet mushroom (Flammulina velutipes), mulberry igneous (Sanghuangporus sp.), Coprinus comatus (Copyindscomatus), red bamboo fungus (Dictyophora rubrovalvata).

[0008] Furthermore, in the above step 2, the amount of the fruiting body or mycelium and the amount of Chelex-100 sodium form added are in a ratio of 1:1.

[0009] Furthermore, the boiling water bath time in the above step 5 is 3-10 minutes.

[0010] Furthermore, the ice bath time in the above step 6 is 1-5 min.

[0011] Furthermore, in the above step 7, the centrifugation time is 1-5 min, and the centrifugal speed is 12000 r / min.

[0012] Beneficial effects of the present invention: Compared with the prior art, the present invention has the following effects: 1) Fewer extraction steps, saving time and effort, verified by experiments: While ensuring the quality of extraction, Extraction can be completed within 20 minutes (conventional CTAB and kit DNA extraction takes at least 3 hours), and as fast as 15 minutes, with high DNA extraction efficiency and significant results. The extracted DNA can be directly used for further research such as ITS amplification sequencing, resolving the common problems of conventional methods such as cumbersome operation procedures, long time consumption, and high costs. 2) The method is simple to operate, highly feasible, and low-cost. Only two reagents need to be purchased: Chelex-100 sodium form and 10×EX Taq buffer. The traditional CTAB method requires: chloroform-isoamyl alcohol (toxic), anhydrous ethanol, EDTA (ethylenediaminetetraacetic acid), Tris-HCl buffer (pH 8.0), β-mercaptoethanol, etc.

[0013] 3) DNA extraction requires little material, and both fruiting bodies (fresh or dried) and mycelium can be quickly extracted. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Flowchart of a rapid and convenient method for extracting large quantities of fungal DNA; Figure 2 Electropherogram of total DNA extracted for a fast and convenient method of extracting fungal DNA in large quantities; Figure 3 The electrophoresis diagram of the amplified ITS band; Figure 4 This is the peak diagram of the sequencing results of the PCR product of Grifola frondosa; Figure 5 This is the peak diagram of sequencing results of PCR products of Ilex strychnifolia; Figure 6 This is the peak diagram of the sequencing results of the PCR product of Lactarius; Figure 7 Flowchart for fungal DNA extraction using the CTAB method.

[0015] Figure 3Middle: On the right is Marker2000, 1 green mold, 2 Stropharia officinalis, 3 Ganoderma lucidum, 4 Grifola frondosa, 5 Oyster mushroom, 6 Morel, 7 Lactarius, 8 Shiitake mushroom, 9 Sugoboshi, 10 Phellinus igniarius, 11 Tricholoma australis (fruiting body), 12 Dictyophora rubra, 13 Auricularia auricula, 14 Hydrangea, 15 Enoki mushroom, 16 Coprinus comatus. DETAILED DESCRIPTION

[0016] Example 1: Figures 1 to 6 As shown, a rapid and convenient method for extracting fungal DNA in large quantities includes the following steps: Step 1: Take a set amount of fungal hyphae or fruiting bodies and place them in a 1.5 mL centrifuge tube; The mycelium or fruiting body is selected from the group consisting of: green mold (Trichoderma sp.), big ball-cap mushroom (Stropharia rugosoannulata), Ganoderma lucidum (Ganoderma lucidum), brown gray mushroom fruiting body (Tricholoma terreum), shiitake mushroom (Lentinula edodes), hairy wood ear (Auricularia polytricha), oyster mushroom (Pleurotus ostreatus), morel (Morchella esculenta (L.) Pers.), milk mushroom fruiting body (Lactarius sp.), maitake (Grifola frondosa), sticky cap milk boletus (Suillus bovinus), pig morel (Panus gigianteus), enoki mushroom (Flammulina velutipes), mulberry igneous (Sanghuangporus sp.), Coprinus comatus (Copyindscomatus), red bamboo fungus (Dictyophora rubrovalvata); Step 2. Add a predetermined amount of Chelex-100 sodium form (Coolaber, model: CC3631) to the centrifuge tube prepared in step 1. The ratio of the amount of fruiting bodies or mycelium to the amount of Chelex-100 sodium form added should be 1:1. Step 3: Add 10-50 µL of sterile deionized water to the centrifuge tube in step 2 and crush the mycelium or fruiting body with a grinding rod provided with a 1.5 mL centrifuge tube. Step 4. Add 10-50µL of 10×EX Taq buffer to the centrifuge tube in step 3 and mix thoroughly on a mixer. Step 5: Place the centrifuge tube after mixing the solution in step 4 in a boiling water bath for 3-10 minutes; Step 6: Place the centrifuge tube after the boiling water bath in step 5 in an ice bath for 1-5 minutes; Step 7: Centrifuge the centrifuge tube after ice bath in step 6 for 1-5 minutes at a centrifugal speed of 12000 r / min; Step 8: Pipette the supernatant (containing DNA) from the solution after centrifugation in step 7 into a new centrifuge tube and store it at -20°C.

[0017] The reagents used were chelating resin (Chelex-100 sodium form), 10×EX Taq buffer, and sterile ddH0.

[0018] Using the method of the present invention, the extraction time of a single sample can be controlled within 20 minutes, while using conventional CTAB and a kit for DNA extraction, the extraction time is at least 3 hours. Using this method, fungal DNA can be quickly extracted and is fully suitable for subsequent PCR amplification experiments.

[0019] DNA quality was tested using ITS-PCR amplification: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used as primers, and PCR amplification was performed using the extracted DNA as a template. The reaction procedure was as follows: denaturation at 95°C for 2 minutes, annealing at 54°C for 30 seconds, extension at 72°C for 1 minute, 35 cycles, and extension at 72°C for 10 minutes, followed by a 4°C reaction stop. The reaction volume was 25 µL. Detection by 1% agarose gel electrophoresis resulted in the following results: Figure 2 As shown in Figure 2, the PCR product fragments obtained were between 500bp and 700bp, indicating that the extracted DNA had good integrity and few impurities, which was consistent with the expected results. The DNA was then sent to the company for sequencing. Figure 4-6 The sequence results are good, the sequencing peak diagram is good, and there are no overlapping peaks, double peaks, or mixed peaks. This shows that the DNA extracted by this DNA extraction method is of good quality, indicating that the DNA extracted by this method can be used for subsequent molecular biology experiments such as PCR amplification.

[0020] Through the above description of the embodiments in combination with the accompanying drawings, technical personnel in the relevant field can understand that for the convenience and brevity of description, only the division of the above-mentioned functional modules is used as an example. In actual applications, the required measurement parameters and objects can be adjusted as needed. The proposed invention has a certain degree of versatility.

Claims

1. A rapid and convenient method for extracting fungal DNA in large quantities, characterized in that: The following steps are involved: Step 1: Take a set amount of fungal hyphae or fruiting bodies and place them in a 1.5 mL centrifuge tube; Step 2: Add a predetermined amount of Chelex-100 sodium form to the centrifuge tube in step 1. Step 3: Add 10-50 µL of sterile deionized water to the centrifuge tube in step 2 and crush the mycelium or fruiting body with a grinding rod provided with a 1.5 mL centrifuge tube. Step 4. Add 10-50µL of 10×EX Taq buffer to the centrifuge tube in step 3 and mix thoroughly on a mixer. Step 5, place the centrifuge tube after mixing the solution in step 4 into a boiling water bath; Step 6: Place the centrifuge tube after the boiling water bath in step 5 in an ice bath; Step 7, centrifuging the centrifuge tube after ice bath in step 6; Step 8: After centrifugation in step 7, remove the supernatant from the solution into a new centrifuge tube and store it at -20°C.

2. A rapid and convenient method for extracting fungal DNA in large quantities according to claim 1, characterized in that: The mycelium or fruiting body in the step 1 is selected from: green mold, giant puffball, ganoderma, brown gray mushroom fruiting body, shiitake mushroom, hairy wood ear, oyster mushroom, morel, milk mushroom fruiting body, maitake mushroom, sticky cap boletus, morel, enoki mushroom, mulberry, coprinus comatus, red stem bamboo fungus.

3. A rapid and convenient method for extracting fungal DNA in large quantities according to claim 1, characterized in that: In step 2, the volume ratio of the amount of fruiting bodies or mycelium to the amount of Chelex-100 sodium form added is 1:

1.

4. A rapid and convenient method for extracting fungal DNA in large quantities according to claim 1, characterized in that: The boiling water bath time in step 5 is 3-10 min.

5. A rapid and convenient method for extracting fungal DNA in large quantities according to claim 1, characterized in that: The ice bath time in step 6 is 1-5 min.

6. A rapid and convenient method for extracting fungal DNA in large quantities according to claim 1, characterized in that: In step 7, the centrifugation time is 1-5 min, and the centrifugal speed is 12000 r / min.

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