Selective culture medium of fusarium and application of selective culture medium in aquatic product detection
By selectively culturing Fusarium with selective culture medium and judging infection based on colony morphological characteristics, the existing detection methods and equipment are solved, and simple and highly accurate Fusarium detection is achieved at the aquatic product aquaculture site.
Patent Information
- Application Number
- CN202510143859.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-10
- Publication Date
- 2025-04-25
AI Technical Summary
The existing Fusarium detection methods have high equipment demand, complex operation and large errors, making it difficult to effectively conduct inspections at the breeding site.
A selective culture medium, including nitrogen-free culture medium, streptomycin, chloramphenicol and cephalomycin, is provided to selectively cultivate Fusarium and determine whether Fusarium is infected with Fusarium based on the morphological characteristics of the colony.
This method is simple, has strong operability, and is highly accurate. It does not require advanced equipment and professional technical personnel to effectively detect Fusarium at the breeding site.
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Abstract
Description
Technical Field
[0001] The present application relates to the technical field related to microbial detection, and specifically provides a selective culture medium for Fusarium and its use in aquatic product detection. Background Art
[0002] Fusarium disease is a common disease in aquaculture, especially in shrimp farming, and is considered the "number one killer" of farmed shrimp.
[0003] Fusarium grows quickly and can form colonies visible to the naked eye within a few days. The outbreak time is usually closely related to high temperature, high-density farming, poor water quality and improper feeding and management. There are many types of Fusarium, with more than 500 species reported so far. Some Fusarium can produce mycotoxins such as fumonisin, deoxynivalenol and zearalenone.
[0004] In recent years, studies have found that the harm of Fusarium to aquatic animals is becoming increasingly serious. Fusarium is widely present in water bodies, and aquatic animals are easily exposed to Fusarium. Fusarium spores or hyphae first need to attach to the body surface of aquatic animals, especially damaged skin or gills. Then they penetrate the damaged skin or gills mechanically, grow rapidly after invasion, form a hyphal network, further expand to the surrounding tissues and secrete a variety of enzymes to provide nutrients for the fungus. After the fungus takes root in the body of aquatic animals, it will produce various toxins, such as deoxynivalenol (DON) and zearalenone (ZEA). These toxins can directly damage the host's tissues, affect its physiological functions, and even cause death. It can also suppress the host's immune system by secreting effector proteins or other mechanisms, reduce its defense capabilities, and cause tissue damage, such as skin ulcers and fin rot, invade internal organs, and finally cause damage to organs such as the liver and kidneys.
[0005] In recent years, with the rapid development of molecular biology, the DNA of Fusarium in aquatic animals can be detected by designing specific primers. This is a sensitive and specific detection method. However, this method requires high equipment requirements and professional technicians to operate, which is not conducive to field detection in aquaculture. Microscopic observation method can be used to examine body tissues and identify the presence of Fusarium spores or hyphae, but this method may have certain errors. Summary of the invention
[0006] In view of this, the present application provides a selective culture medium for Fusarium and its use in aquatic product detection. The present application selectively cultivates Fusarium using a selective culture medium, and then determines whether aquatic products are infected with Fusarium based on the morphological characteristics of the colonies. This method is not only simple and easy to operate, but also highly accurate.
[0007] In a first aspect, the present application provides a selective culture medium for selectively culturing Fusarium, the selective culture medium comprising a basal culture medium, streptomycin, chloramphenicol and cephalosporin; the basal culture medium is selected from a nitrogen-free culture medium; the concentration of the streptomycin is (60-100) μg / mL, the concentration of the chloramphenicol is (10-50) μg / mL, and the concentration of the cephalosporin is (60-100) μg / mL.
[0008] In some optional embodiments, the concentration of streptomycin is (70-90) μg / mL; for example, 70 μg / mL, 71 μg / mL, 72 μg / mL, 73 μg / mL, 74 μg / mL, 75 μg / mL, 76 μg / mL, 77 μg / mL, 78 μg / mL, 79 μg / mL, 80 μg / mL, 81 μg / mL, 82 μg / mL, 83 μg / mL, 84 μg / mL, 85 μg / mL, 86 μg / mL, 87 μg / mL, 88 μg / mL, 89 μg / mL or 90 μg / mL, etc. In some specific embodiments, the concentration of streptomycin is 80 μg / mL.
[0009] In some optional embodiments, the concentration of chloramphenicol is (20-40) μg / mL; for example, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 26 μg / mL, 27 μg / mL, 28 μg / mL, 29 μg / mL, 30 μg / mL, 31 μg / mL, 32 μg / mL, 33 μg / mL, 34 μg / mL, 35 μg / mL, 36 μg / mL, 37 μg / mL, 38 μg / mL, 39 μg / mL or 40 μg / mL, etc. In some specific embodiments, the concentration of chloramphenicol is 30 μg / mL.
[0010] In some optional embodiments, the concentration of the cephalosporin is (70-90) μg / mL; for example, 70 μg / mL, 71 μg / mL, 72 μg / mL, 73 μg / mL, 74 μg / mL, 75 μg / mL, 76 μg / mL, 77 μg / mL, 78 μg / mL, 79 μg / mL, 80 μg / mL, 81 μg / mL, 82 μg / mL, 83 μg / mL, 84 μg / mL, 85 μg / mL, 86 μg / mL, 87 μg / mL, 88 μg / mL, 89 μg / mL or 90 μg / mL, etc. In some specific embodiments, the concentration of the cephalosporin is 80 μg / mL.
[0011] In some specific embodiments, the concentration of streptomycin is 80 μg / mL, the concentration of chloramphenicol is 30 μg / mL, and the concentration of cephalosporin is 80 μg / mL.
[0012] In some optional embodiments, based on the nitrogen-free medium, the nitrogen-free medium includes a carbon source (0.7-1.3) wt% (e.g., 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.2 wt% or 1.3 wt%, etc.), dipotassium hydrogen phosphate trihydrate (0.2-0.6) wt% (e.g., 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt% or 0.6 wt%, etc.), potassium dihydrogen phosphate (0-0.4) wt% (e.g., 0 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt% or 0.4 wt%, etc.), magnesium sulfate heptahydrate (0.2-0.6) wt% (e.g., 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt% or 0.6wt%, etc.), calcium chloride (0.15-0.35)wt% (e.g. 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt% or 0.35wt%, etc.), calcium carbonate (0.2-0.6)wt% (e.g. 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt% or 0.6wt%, etc.), agar powder (1.2-1.8)wt% (e.g. 1.2wt%, 1.3wt%, 1.4wt%, 1.5wt%, 1.6wt%, 1.7wt% or 1.8wt%, etc.), and the balance is water.
[0013] In some optional embodiments, the carbon source is selected from at least one of sucrose, glucose and galactose. In some specific embodiments, the carbon source is selected from sucrose.
[0014] In some specific embodiments, based on the nitrogen-free culture medium, the nitrogen-free culture medium includes 1 wt % sucrose, 0.4 wt % dipotassium hydrogen phosphate trihydrate, 0 wt % potassium dihydrogen phosphate, 0.5 wt % magnesium sulfate heptahydrate, 0.25 wt % calcium chloride, 0.4 wt % calcium carbonate, 1.5 wt % agar powder, and the balance is water.
[0015] In some specific embodiments, based on the nitrogen-free culture medium, the nitrogen-free culture medium includes 1 wt% sucrose, 0.4 wt% dipotassium hydrogen phosphate trihydrate, 0.2 wt% potassium dihydrogen phosphate, 0.5 wt% magnesium sulfate heptahydrate, 0.25 wt% calcium chloride, 0.4 wt% calcium carbonate, 1.5 wt% agar powder, and the balance is water.
[0016] In some specific embodiments, based on the nitrogen-free culture medium, the nitrogen-free culture medium includes 1 wt % sucrose, 0.4 wt % dipotassium hydrogen phosphate trihydrate, 0.4 wt % potassium dihydrogen phosphate, 0.5 wt % magnesium sulfate heptahydrate, 0.25 wt % calcium chloride, 0.4 wt % calcium carbonate, 1.5 wt % agar powder, and the balance is water.
[0017] In some optional embodiments, the Fusarium is selected from at least one of Fusarium nivalis, Fusarium moniliformis, Fusarium graminearum, Fusarium trilineata, Fusarium pyriformis, Fusarium pseudosporum, Fusarium equiseti, Fusarium solani and Fusarium oxysporum. In other optional embodiments, the Fusarium is selected from at least one of Fusarium nivalis, Fusarium moniliformis and Fusarium graminearum.
[0018] In a second aspect, the present application provides a use of the selective culture medium described in the first aspect in aquatic product detection.
[0019] In some embodiments, the aquatic product is selected from shrimp (Penaeus orientalis).
[0020] In some specific embodiments, the test items for the aquatic product test are selected from Fusarium detection.
[0021] In a third aspect, the present application provides a method for detecting Fusarium in aquatic products, the method comprising the following steps: Step S1, preparing aquatic products into samples to be tested; Step S2, inoculating the sample to be tested in the selective culture medium described in the first aspect to obtain colonies; Step S3: judging whether the aquatic product is infected with Fusarium according to the morphological characteristics of the spores in the bacterial colony.
[0022] In some specific embodiments, in step S1, the aquatic product is selected from prawns. Further, the operation process of step S1 is: the gill filaments and cephalothorax of the prawns are mixed and ground with sterile water to obtain a sample to be tested.
[0023] In some optional embodiments, in step S2, the culture temperature is (20-40)°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.); and / or, the culture time is (36-84) hours (e.g., 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours or 84 hours, etc.). In some specific embodiments, the culture temperature is 28°C.
[0024] In some optional embodiments, in step S3, the morphological characteristics of spores in the colony are observed under a microscope to determine whether the aquatic product is infected with Fusarium.
[0025] This application has the following beneficial effects: First, the present application utilizes a selective culture medium to selectively culture Fusarium, and then determines whether the aquatic product is infected with Fusarium based on the morphological characteristics of the colonies. This method is not only simple and easy to operate, but also highly accurate.
[0026] Second, the Fusarium in the experimental sample cannot grow normally in the basic culture medium containing nitrogen source, which indicates that the basic culture medium containing nitrogen source cannot be used for selective cultivation of Fusarium. Furthermore, the addition of potassium dihydrogen phosphate in the nitrogen-free culture medium and the amount of addition can further affect the accuracy of the selective culture medium of the present application.
[0027] Third, the combination of "streptomycin, chloramphenicol and cephalosporin" used in this application can significantly improve the accuracy of the selective culture medium of this application. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 These are the results of culturing Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum in the selective culture medium of Preparation Example 2.
[0029] Figure 2 These are the culture results of Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum in the selective culture medium of Comparative Example 2.
[0030] Figure 3 These are the culture results of Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum in the selective culture medium of Comparative Example 4. DETAILED DESCRIPTION
[0031] The present application discloses a selective culture medium for Fusarium and its use in aquatic product detection. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.
[0032] Terminology explanation: The term "selective culture" refers to a type of culture designed based on the special nutritional requirements of a certain microorganism or its resistance to certain chemical or physical factors. It has the function of developing inferior bacteria in a mixed bacteria into superior bacteria, so it is widely used in fields such as strain screening.
[0033] The term "nitrogen-free medium" is a medium lacking any bound inorganic or organic nitrogen source.
[0034] Unless otherwise stated, all reagents used in this application were commercially available.
[0035] Among them, Potato Dextrose Broth (PDB) culture medium was purchased from Kingclone (Beijing) Biotechnology Co., Ltd.
[0036] Potato Dextrose Agar (PDA) medium was purchased from Qingdao Haibo Biotechnology Co., Ltd.; PDA medium: 20 g glucose, 20 g potato, 20 g agar, and 1000 mL distilled water.
[0037] Streptomycin (CAS No. 57-92-1) and cephalosporin (CAS No. 64485-93-4) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; chloramphenicol (CAS No. 56-75-7) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.
[0038] Unless otherwise stated, the "Fusarium nivale" used in this application is deposited in the Henan Industrial Microbiological Strain Engineering Technology Research Center, the deposit address is Hedian Light Industrial Park, Jingangtai Avenue, Shangcheng County, Xinyang City, Henan Province, the deposit date is 2021.12.23, and the deposit number is BNCC337556.
[0039] Unless otherwise stated, the "Fusarium moniliforme" used in this application is deposited in the China Industrial Microbiological Culture Collection Center, the deposit address is Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, the deposit date is 2021.12.14, and the deposit number is CICC2490.
[0040] Unless otherwise stated, the "Fusarium graminearum" used in this application is deposited in the Henan Industrial Microbiological Strain Engineering Technology Research Center, the deposit address is Hedian Light Industrial Park, Jingangtai Avenue, Shangcheng County, Xinyang City, Henan Province, the deposit date is 2021.12.14, and the deposit number is BNCC113713.
[0041] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.
[0042] Preparation of nitrogen-free culture medium :
[0043] Table 1. Ingredients of nitrogen-free culture medium:
[0044] Preparation of selective culture medium :
[0045] Table 2, ingredients of selective culture medium of Preparation Example 1 and Comparative Examples 1 to 4:
[0046] The method for preparing the selective culture medium comprises the following steps: sterilizing the basic culture medium at high temperature and high pressure at 116°C (specifically 116°C) for 30 minutes; when the basic culture medium is cooled to below 50°C, adding streptomycin, chloramphenicol and cephalosporin to obtain a selective culture medium.
[0047] Cultivation of Fusarium :
[0048] Select Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum for cultivation. Specific cultivation method: Inoculate Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum into 5 mL of PDB medium respectively, and culture at 28°C and 150 rpm for 24 hours, aspirate 1 mL of fermentation liquid and place it in a 2 mL centrifuge tube, shake it thoroughly to obtain a spore suspension. Aspirate 2 μL of the spore suspension of each Fusarium and inoculate it into the same selective culture medium, incubate it at 28°C for 2 to 3 days, and observe whether Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum can grow normally in the selective culture medium. The observation results are as follows: Figures 1 to 3 shown.
[0049] from Figures 1 to 3 It can be seen that Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum can all grow normally in the selective culture medium of Preparation Example 2, Preparation Comparative Example 2 and Preparation Comparative Example 4, but the colony sizes are slightly different.
[0050] Methods and validation for detecting Fusarium in aquatic products :
[0051] The method for detecting Fusarium in aquatic products specifically comprises the following steps: Step S1, selecting shrimp infected with Fusarium wilt as experimental samples, mixing and grinding the gill filaments and cephalothorax of the shrimp with sterile water to obtain a sample to be tested; Step S2, taking 100 μL of the test solution and applying it to a selective culture medium, culturing at 28° C. for 60 hours to obtain colonies; Step S3: observing the morphological characteristics of spores in the colony under a microscope to determine whether the aquatic product is infected with Fusarium.
[0052] Verification of Fusarium: white hyphae in the colony were picked and inoculated into PDB medium for culture; the fungus was collected and DNA was extracted using a fungal genomic DNA extraction kit (model R33123, purchased from Shanghai Yuanye Biotechnology Co., Ltd.); based on the internal transcribed spacer (ITS) amplicon sequencing method, the colony was identified as a Fusarium colony.
[0053] Amplification system: 1 μL DNA template, 25 μL Taq enzyme, 1 μL primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), 1 μL primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and make up to 50 μL with sterile water.
[0054] Amplification program: pre-denaturation at 95°C for 3 min, (denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min) 30 cycles, extension for 5 min, and storage at 4°C.
[0055] Sequencing method: Sanger sequencing.
[0056] Table 3. Selective culture results of Fusarium in experimental samples and identification results of ITS amplicon sequencing method:
[0057] As can be seen from Table 3, the present application utilizes a selective culture medium to selectively culture Fusarium, and then determines whether the experimental sample contains Fusarium based on the morphological characteristics of the colonies. This method is not only simple and easy to operate, but also highly accurate.
[0058] By comparing Preparation Examples 1 to 3 and Comparative Preparation Example 4, it can be seen that the Fusarium in the experimental sample cannot grow normally in the basic culture medium containing a nitrogen source; thus, it is shown that the basic culture medium containing a nitrogen source cannot be used for selective cultivation of Fusarium. Further, by comparing Examples 1 to 3, it can be seen that the addition of potassium dihydrogen phosphate in the nitrogen-free culture medium and the amount of addition can further affect the accuracy of the selective culture medium of the present application.
[0059] By comparing Preparation Example 2 with Comparative Preparation Examples 1 to 3, it can be seen that the combination of "streptomycin, chloramphenicol and cephalosporin" used in the present application can significantly improve the accuracy of the selective culture medium of the present application.
[0060] The above is a detailed introduction to a selective culture medium for Fusarium provided by the present application and its use in aquatic product detection. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.
Claims
1. A selective culture medium for Fusarium, characterized in that The selective culture medium comprises a basic culture medium, streptomycin, chloramphenicol and cephalosporin; the basic culture medium is selected from a nitrogen-free culture medium; the concentration of the streptomycin is (60-100) μg / mL, the concentration of the chloramphenicol is (10-50) μg / mL, and the concentration of the cephalosporin is (60-100) μg / mL.
2. The selective culture medium according to claim 1, characterized in that The concentration of streptomycin is (70-90) μg / mL; Optionally, the concentration of streptomycin is 80 μg / mL.
3. The selective culture medium according to claim 1, characterized in that The concentration of chloramphenicol is (20-40) μg / mL; Optionally, the concentration of chloramphenicol is 30 μg / mL.
4. The selective culture medium according to claim 1, characterized in that The concentration of the cephalosporin is (70-90) μg / mL; Optionally, the concentration of the cephalosporin is 80 μg / mL.
5. The selective culture medium according to claim 1, characterized in that Taking the nitrogen-free culture medium as a benchmark, the nitrogen-free culture medium includes a carbon source (0.7-1.3) wt%, dipotassium hydrogen phosphate trihydrate (0.2-0.6) wt%, potassium dihydrogen phosphate (0-0.4) wt%, magnesium sulfate heptahydrate (0.2-0.6) wt%, calcium chloride (0.15-0.35) wt%, calcium carbonate (0.2-0.6) wt%, agar powder (1.2-1.8) wt%, and the balance is water.
6. The selective culture medium according to claim 5, characterized in that The carbon source is selected from at least one of sucrose, glucose and galactose; Optionally, the carbon source is selected from sucrose.
7. The selective culture medium according to claim 1, characterized in that The Fusarium is selected from at least one of Fusarium nivalis, Fusarium moniliforme, Fusarium graminearum, Fusarium trilineatum, Fusarium pyrifolium, Fusarium pseudosporum, Fusarium equisetum, Fusarium solani, and Fusarium oxysporum; Optionally, the Fusarium is selected from at least one of Fusarium nivalis, Fusarium moniliforme and Fusarium graminearum.
8. Use of the selective culture medium according to any one of claims 1 to 7 in the detection of aquatic products.
9. A method for detecting Fusarium in aquatic products, characterized in that: It includes the following steps: Step S1, preparing aquatic products into samples to be tested; Step S2, inoculating the sample to be tested in the selective culture medium as claimed in any one of claims 1 to 7 to obtain colonies; Step S3: judging whether the aquatic product is infected with Fusarium according to the morphological characteristics of the spores in the bacterial colony.
10. The method according to claim 9, characterized in that The culture temperature is (20-40)°C; Optionally, the culturing time is (36-84) hours.