A method for isolating Aspergillus fumigatus
Through the method of mixing liquid Martin's culture medium with soil suspension and high-temperature culture, the problem of low separation efficiency of Aspergillus fumigatum in the soil is solved, efficient and accurate separation of Aspergillus fumigatum is achieved, and a large amount of germplasm resources are provided.
Patent Information
- Application Number
- CN202310120842.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-16
- Publication Date
- 2025-08-12
- Estimated Expiration
- 2043-02-16
AI Technical Summary
In the prior art, the efficiency of isolating Aspergillus fumigatum from soil is low. It is mainly due to the small contact area between the culture medium and the soil and the unsuitable temperature, which leads to excessive growth of other dominant fungi, making it difficult to efficiently isolate Aspergillus fumigatum strains.
Martin's culture medium in mixed liquid state was mixed with soil suspension and solidified, and a layer of Martin's culture medium was added to the liquid state, and cultured at 48°C. The high-temperature resistance characteristics of Aspergillus fumigatum fumigatum was used to inhibit the growth of other fungi. Colonies with fleece or flocculent shape and green center white edges were selected as the target Aspergillus fumigatum.
The separation efficiency of Aspergillus fumigatum is significantly improved. A mixed bacterial plate can obtain 5 to 8 colonies, the separation efficiency is several times improved, and the identification accuracy reaches 100%, providing rich germplasm resources for subsequent research.
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Figure CN116254317B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial separation, and particularly relates to a method for separating Aspergillus fumigatus. Background Art
[0002] The soil environment contains a variety of nutritional conditions suitable for microbial growth and serves as a natural medium for microbial growth and reproduction. Isolating microorganisms from soil is an important way to obtain microbial germplasm resources. Aspergillus fumigatus is a fungus with a wide distribution in ecological niches around the world. It mainly engages in saprophytic and parasitic activities and is a major opportunistic pathogen in humans. It can infect the lungs of immunocompromised or immunodeficient individuals and cause fatal aspergillosis. Reports have found that resistant strains of Aspergillus fumigatus can still be isolated from patients with aspergillosis who do not use triazole drugs, suggesting that the resistant strains may originate from the environment. Therefore, the efficient isolation of Aspergillus fumigatus strains from the soil environment is conducive to research in areas such as Aspergillus genetic diversity, environmental adaptability, genetic evolution, drug resistance prevalence monitoring, GWAS technology screening of candidate resistance genes, and germplasm resource development and utilization.
[0003] Currently, the main methods for isolating Aspergillus fumigatus from soil are the dilution spread plate method and the dilution mixing plate method. Foreign countries have also used a small amount of soil sample to contact culture medium to isolate Aspergillus fumigatus. Although these methods can isolate a certain amount of Aspergillus fumigatus from soil samples, they have the following shortcomings:
[0004] (1) The dilution plate method is to spread different gradients of soil dilution on the surface of the separation culture medium. Because the culture medium absorbs less soil dilution, the amount of soil dilution added is often controlled at about 200 μL. Too much added will lead to problems such as difficulty in drying and easy spread. In addition, the culture temperature is generally controlled at a temperature suitable for the growth of most fungi (about 28°C), which leads to excessive growth of other dominant fungi on the separation culture medium, and the efficiency of separating Aspergillus fumigatus is low. Figures 1-2 As shown, generally only 1 to 2 Aspergillus fumigatus colonies can be obtained.
[0005] (2) The dilution mixing plate method is to mix soil dilutions of different gradients with unsolidified solid culture medium and then culture at a suitable temperature (around 28°C). However, since the soil suspension is diluted and the culture at a suitable temperature also causes excessive growth of other dominant fungi, the efficiency of isolating Aspergillus fumigatus is also low.
[0006] (3) Foreign methods take a small amount of soil sample (about 0.2 g) and place it in a sterile 2.0 ml round-bottom centrifuge tube, and add a certain amount (about 0.5 ml) of culture medium that is about to solidify. Often, only the surface of the soil sample can contact the culture medium. Due to the small contact area, the efficiency of separating Aspergillus fumigatus is also not high. Summary of the Invention
[0007] In view of this, an object of the present invention is to provide a method for isolating Aspergillus fumigatus, which can efficiently isolate Aspergillus fumigatus.
[0008] The present invention provides a method for isolating Aspergillus fumigatus, comprising the following steps:
[0009] The suspension of the soil to be separated and the first portion of the liquid screening culture medium are mixed in a culture dish to obtain a mixture. After the mixture solidifies, the second portion of the liquid screening culture medium is covered on the surface of the solidified mixture until solidified. The mixture is cultured at 48°C for 2-3 days, and the strain with velvety or flocculent colonies, a green center and white edges is selected as the target Aspergillus fumigatus;
[0010] The screening culture medium is based on Martin's culture medium and further contains penicillin and streptomycin.
[0011] Preferably, the concentration of streptomycin in the screening medium is 50-100 μg / ml; the concentration of penicillin in the screening medium is 100-150 μg / ml.
[0012] Preferably, the method for preparing the suspension of soil to be separated comprises the following steps: mixing the soil to be separated and sterile water, and shaking to obtain a suspension of soil to be separated; the mass ratio of the soil to be separated and the sterile water is 1:1.
[0013] Preferably, the oscillation speed is 100-200 r / min; and the oscillation time is 1-3 h.
[0014] Preferably, the volume ratio of the suspension of the soil to be separated to the first portion of the screening culture medium in liquid state is (1-4):20.
[0015] Preferably, the volume ratio of the suspension of the soil to be separated and the second portion of the screening culture medium in liquid state is (1-4):20.
[0016] Preferably, based on a culture dish volume of 90 mL, the amount of the suspension of soil to be separated added to each culture dish is 500-2000 μl.
[0017] Preferably, the temperature of the liquid screening culture medium is 50-52°C.
[0018] The invention provides a method for separating Aspergillus fumigatus. The method comprises mixing a soil suspension with a liquid Martin's medium containing a double antibody, and then adding a layer of the Martin's medium containing the double antibody after solidification. This method avoids scalding of Aspergillus fumigatus spores due to a large amount of medium, a long solidification time, or an excessively high temperature, and enables the Aspergillus fumigatus spores in the soil suspension to fully contact the medium, thereby improving the separation efficiency by several times. Based on a conventional dilution mixing plate method, the method adopts a method in which a soil suspension stock solution is directly mixed with an unsolidified Martin's medium to perform plate mixing. The method is simple and has high separation efficiency. In addition, the present invention utilizes the characteristic of Aspergillus fumigatus to tolerate a certain high temperature and culture the isolation culture medium at 45 to 48 ° C. This temperature condition can inhibit the growth of most fungi, while Aspergillus fumigatus is not affected. Therefore, after 2 to 3 days of culture, there are fewer colonies of miscellaneous fungi that can grow normally on the culture medium. The morphological characteristics of Aspergillus fumigatus colonies are obvious, that is, the colonies are velvety or flocculent, with a green center and a white edge. Aspergillus fumigatus can be accurately identified based on the morphological characteristics. At the same time, the separation efficiency is greatly improved, and 5 to 8 colonies can be obtained from a mixed bacterial plate. At present, more than 1,200 strains of Aspergillus fumigatus have been isolated from soil samples by the method of the present invention, and the accuracy rate of ITS identification has reached 100%. The method of the present invention greatly improves the isolation efficiency of Aspergillus fumigatus in soil environments, and provides a large amount of germplasm resources for subsequent research in the fields of genetic diversity, environmental adaptability, genetic evolution, drug resistance epidemic monitoring, GWAS technology screening of drug resistance candidate genes, and germplasm resource development and utilization. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Martin's medium was used, and the plate dilution method (10 -3 , 10 -4 Comparison of the effects of separating Aspergillus fumigatus from soil samples using the plate mixed culture method and different temperature conditions;
[0020] Figure 2 PDA medium was used, and the plate dilution method (10 -3 , 10 -4 The comparison of the effects of the plate mixed culture method and the Aspergillus fumigatus method in isolating soil samples under different temperature conditions. DETAILED DESCRIPTION
[0021] The present invention provides a method for isolating Aspergillus fumigatus, comprising the following steps:
[0022] The suspension of the soil to be separated and the first portion of the liquid screening culture medium are mixed in a culture dish to obtain a mixture. After the mixture solidifies, the second portion of the liquid screening culture medium is covered on the surface of the solidified mixture until solidified. The mixture is cultured at 48°C for 2-3 days, and the strain with velvety or flocculent colonies, a green center and white edges is selected as the target Aspergillus fumigatus;
[0023] The screening culture medium is based on Martin's culture medium and further contains penicillin and streptomycin.
[0024] In the present invention, the Martin's medium uses water as a solvent and includes the following components in the following concentrations: 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 5.0 g of peptone, 10.0 g of glucose, and 20.0 g of agar; preferably, the Martin's medium uses water as a solvent and is composed of the following components in the following concentrations: 1.0 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate heptahydrate, 5.0 g of peptone, 10.0 g of glucose, and 20.0 g of agar; the pH value of the Martin's medium is preferably 6 to 8, more preferably 6.5 to 7.5, and most preferably 7.
[0025] In the present invention, the concentration of streptomycin in the screening medium is preferably 50-100 μg / ml, more preferably 80 μg / ml; the concentration of penicillin in the screening medium is preferably 100-150 μg / ml, more preferably 120 μg / ml. In the present invention, the functions of the streptomycin and penicillin are to prevent bacterial contamination and ensure the isolation of the target strain.
[0026] The present invention mixes a soil suspension with a liquid Martin's medium containing double antibodies, and after solidification, adds a layer of Martin's medium containing double antibodies. This not only avoids the scalding death of Aspergillus fumigatus spores due to a large amount of culture medium, a long solidification time, and an excessively high temperature, but also enables the Aspergillus fumigatus spores in the soil suspension to fully contact the culture medium, thereby improving the separation efficiency by several times.
[0027] In the present invention, the method for preparing the suspension of soil to be separated preferably comprises the following steps: mixing the soil to be separated with sterile water and shaking to obtain a suspension of soil to be separated; the mass ratio of the soil to be separated to the sterile water is preferably 1:1. The present invention has low requirements for soil samples and does not require prior drying, sieving, or grinding of the soil sample, thereby avoiding sample contamination caused by prior processing.
[0028] The present invention utilizes the characteristic of Aspergillus fumigatus being able to tolerate a certain high temperature, and cultivates the separation culture medium at 45-48°C. The temperature condition can inhibit the growth of most fungi, but Aspergillus fumigatus is not affected. Therefore, after culturing for 2-3 days, fewer miscellaneous fungal colonies can grow normally on the culture medium, and the Aspergillus fumigatus colony has obvious morphological characteristics, that is, the colony is velvety or flocculent, with a green center and a white edge. Aspergillus fumigatus can be accurately identified based on the morphological characteristics, and the separation efficiency is greatly improved. 5-8 colonies can be obtained from one mixed bacteria plate.
[0029] In the present invention, the rotation speed of the oscillation is preferably 100 to 200 r / min, more preferably 150 to 180 r / min; the oscillation time is preferably 1 to 3 hours, more preferably 2 hours.
[0030] In the present invention, the volume ratio of the suspension of the soil to be separated to the first portion of the liquid screening culture medium is preferably (1-4):20, more preferably (2-3):20.
[0031] In the present invention, the volume ratio of the suspension of the soil to be separated and the second portion of the screening culture medium in liquid state is preferably (1-4):20, more preferably (2-3):20.
[0032] In the present invention, based on a volume of 90 mL of a culture dish, the amount of the suspension of soil to be separated added to each culture dish is preferably 500 to 2000 μl, more preferably 1000 to 1500 μl.
[0033] In the present invention, the temperature of the liquid screening medium is preferably 50-52°C.
[0034] The technical solutions of the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention.
[0035] Example 1
[0036] 1. Treat the soil
[0037] The control example used the plate dilution method: under sterile conditions, 10 g of soil from the target plot was placed in a flask containing 90 ml of sterile water. The flask was sealed and shaken on a shaker at 100 rpm for about 2 hours to prepare a soil suspension.
[0038] The test example uses the plate mixing method: under sterile conditions, take 25g of soil sample collected from the target sample site and place it in a triangular flask containing 25ml of sterile water. After the triangular flask is sealed, it is placed on a shaker at 100r / min for 2h to prepare a soil suspension;
[0039] 2. Prepare culture medium
[0040] Martin's medium: Weigh 1.0 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 5.0 g peptone, 10.0 g glucose, and 20.0 g agar, add water to make up to 1000 ml, divide into Erlenmeyer flasks, seal the flasks, and place in an autoclave at 121°C for 30 min.
[0041] Prepare PDA culture medium: weigh 200 g of peeled potatoes, 20 g of glucose, and 20 g of agar, add water to make up to 1000 ml, divide into Erlenmeyer flasks, seal them, and place them in an autoclave at 121°C for 30 min.
[0042] 3. Preparation of dual-antibody culture medium
[0043] After sterilization, the Martin's and PDA culture media were placed in a constant temperature water bath at 52°C to keep them in a liquid state. After the temperature of the culture medium became constant, the culture medium was removed and double antibiotics (streptomycin and penicillin) were added under sterile conditions to make the final concentrations of streptomycin and penicillin in the culture medium 50 μg / ml and 100 μg / ml, respectively.
[0044] 4. Isolation of Aspergillus fumigatus
[0045] Plate dilution method: under sterile conditions, dilute the soil suspension to 10 -3 and 10 -4 concentration, use a pipette to draw 200 μl of 10 -3 and 10 -4 The diluted solution of the concentration was added to 90 mm Martin's and PDA solid culture dishes containing double antibodies, spread evenly with a sterile coating rod, and placed inverted in a constant temperature incubator at 28°C, 35°C, 48°C and 55°C for 2 to 3 days.
[0046] Plate mixing method: Gently shake the prepared soil suspension to mix it, use a pipette to draw 500μl of soil suspension into a 90mm sterile culture dish, then pour in about 10ml of 52℃ Martin's medium with double antibodies (PDA medium with the same conditions is used as a reference), gently rotate the culture dish horizontally to mix the soil suspension with the Martin's medium containing double antibodies and PDA medium, and after solidification, pour about 10ml of Martin's medium containing double antibodies and PDA medium into the culture dish again. After solidification, seal it and place it in a constant temperature incubator at 28℃, 35℃, 48℃ and 55℃ for inverted culture for 2-3 days. During this period, do not check the growth status of the colony in the culture dish frequently to prevent Aspergillus fumigatus spores from falling into the culture dish.
[0047] 5. Identification of Aspergillus fumigatus
[0048] (1) Morphological identification
[0049] Observe the morphology of the colonies on the culture dish. The colonies are velvety or flocculent, with a green center and white edges. Figure 1 and Figure 2, culture temperatures of 28°C, 35°C, 48°C, and 55°C, respectively) are candidate target strains. By comparing the results of different treatments, it was found that the patented method can obtain 5 to 8 colonies per mixed plate, which is superior to the plate dilution method and PDA culture medium as a reference. Use a sterile inoculation needle to pick a small amount of mycelium and transfer it to a new PAD solid culture medium containing double antibodies. After sealing, place it in an inverted culture at 48°C. After 4 days, scrape a small amount of mycelium into a 20% glycerol tube and store it in a -80°C ultra-low temperature freezer. The remaining mycelium is used for DNA extraction.
[0050] (2) Molecular biological identification
[0051] First, the DNA of the candidate target strain was extracted using the improved CTAB method (Zhang et al. 2016), and then PCR amplification was performed using the universal fungal identification primers ITS4 and ITS5 (White et al. 1990). The amplified products were sent to a sequencing company for bidirectional sequencing. After the sequencing sequences were spliced, sequence similarity comparison and analysis were performed using the Blast program on the NCBI official website. If the ITS sequence similarity between the candidate target strain and the Aspergillus fumigatus strain in the NCBI database was above 98%, the strain could be considered to be Aspergillus fumigatus.
[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention rather than all the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A method for isolating Aspergillus fumigatus, comprising the following steps: The suspension of the soil to be separated and the first portion of the liquid screening culture medium are mixed in a culture dish to obtain a mixture. After the mixture solidifies, the second portion of the liquid screening culture medium is covered on the surface of the solidified mixture until solidified. The mixture is cultured at 48°C for 2-3 days, and the strain with velvety or flocculent colonies, a green center and white edges is selected as the target Aspergillus fumigatus; The screening culture medium is based on Martin's culture medium and further contains penicillin and streptomycin.
2. The separation method according to claim 1, characterized in that The concentration of streptomycin in the screening culture medium is 50-100 μg / ml; the concentration of penicillin in the screening culture medium is 100-150 μg / ml.
3. The separation method according to claim 1, characterized in that The method for preparing the suspension of the soil to be separated comprises the following steps: mixing the soil to be separated and sterile water, and shaking to obtain the suspension of the soil to be separated; the mass ratio of the soil to be separated and the sterile water is 1:
1.
4. The separation method according to claim 3, characterized in that The rotation speed of the oscillation is 100 to 200 r / min; and the oscillation time is 1 to 3 hours.
5. The separation method according to claim 1, characterized in that The volume ratio of the suspension of the soil to be separated to the first part of the screening culture medium in a liquid state is (1-4):
20.
6. The separation method according to claim 1, characterized in that The volume ratio of the suspension of the soil to be separated and the second part of the screening culture medium in a liquid state is (1-4):
20.
7. The separation method according to claim 5 or 6, characterized in that Assuming the volume of the culture dish is 90 mL, the amount of the suspension of soil to be separated added to each culture dish is 500 to 2000 μl.
8. The separation method according to claim 1, characterized in that The temperature of the liquid screening culture medium is 50-52°C.
Citation Information
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