Method for inducing mycelial phase of Metschnosaccharomyces binapi based on carbon quantum dots and application of method

Abstract

The invention discloses a carbon quantum dot-based method for inducing a mycelial phase of Meikiwifruit microzymes and application thereof. The method comprises the following steps: preparing carbon quantum dots by a hydrothermal method, carrying out mycelial phase induction culture and carrying out morphological observation and confirmation. The carbon quantum dots are prepared by carrying out a hydrothermal reaction on citric acid and urea at 230 DEG C for 15 hours, and the particle size is 2-8 nm; the carbon quantum dots are added into a Sabouraffi glucose culture medium, the final concentration is 0.5 mg / mL, and the Metschkiwifruit microzymes can be obviously induced to form a hypha phase when the Metschkiwifruit microzymes are cultured at 28 DEG C for 24-28 hours; microscopic observation and PCR (Polymerase Chain Reaction) molecule confirmation are combined, so that the hypha formation rate can reach 85% or above. The method disclosed by the invention provides a stable and reliable experimental material for researching the pathogenic mechanism and the prevention and control technology of the Metschwsky microzyme.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aquatic animal pathogenic microorganism research, and particularly relates to a method for inducing Metschnikowia bicuspidata mycelial phase based on carbon quantum dots and application thereof. BACKGROUND

[0002] Metschnikowia bicuspidata is an important pathogenic fungus of Eriocheir sinensis, which can cause "milk disease" of Eriocheir sinensis, resulting in atrophy of hepatopancreas, weight loss and high mortality of crabs.

[0003] Metschnikowia bicuspidata has two forms of yeast phase and mycelial phase, and the formation of mycelial phase is closely related to its pathogenicity. During host infection, the fungus can transform from yeast phase to mycelial phase, thereby enhancing its tissue penetration ability and expression of virulence factors. In-depth study on the formation mechanism of mycelial phase is of great significance for elucidating the pathogenic mechanism of the fungus and developing new prevention strategies.

[0004] Current studies on Metschnikowia bicuspidata show that the pathogen can cause infection of various aquatic cultured animals, including Eriocheir sinensis, Portunus trituberculatus and river crab, which are crustaceans with high economic value, resulting in the occurrence of "milk disease". The diseased crabs show symptoms such as opaque milky white muscle, emulsified blood lymph and weakened body, and eventually die. At present, there is no effective treatment method for the disease.

[0005] Therefore, the study on the pathogenic mechanism of Metschnikowia bicuspidata has important theoretical and practical significance. However, the existing technology has problems such as low induction efficiency and poor reproducibility in the induction method of Metschnikowia bicuspidata mycelial phase. Commonly used induction methods include serum induction method and corn meal medium induction method, but the mycelial formation rate is usually only 40-60%, and the induction conditions are unstable, which makes it difficult to provide sufficient and stable mycelial phase materials for in-depth study of the pathogenic mechanism. SUMMARY

[0006] In view of the above technical problems of low induction efficiency and poor reproducibility of Metschnikowia bicuspidata mycelial phase, the application provides a method for inducing Metschnikowia bicuspidata mycelial phase based on carbon quantum dots and application thereof, which provides stable and reliable experimental materials for in-depth study of the pathogenic mechanism of the fungus.

[0007] As a new type of nanomaterial, the carbon quantum dots have good biocompatibility, fluorescence properties and surface activity, and have broad application prospects in the field of biomedicine. Studies have found that carbon quantum dots can significantly affect the growth and morphological transformation of microorganisms by regulating cell membrane permeability and affecting cell metabolic activity. Therefore, the application induces Metschnikowia bicuspidata mycelial phase based on carbon quantum dots.

[0008] The object of the application is achieved by the following technical solutions: The application discloses a method for inducing hypha phase of Leucosporidium cryptocidum based on carbon quantum dots. a) Preparation of carbon quantum dots by a hydrothermal method: 2.0 g of citric acid and 1.0 g of urea are dissolved in 20 mL of deionized water, and hydrothermal reaction is carried out at 230 DEG C for 15 hours to obtain a carbon quantum dot solution; b) Preparation of an induction medium: the carbon quantum dot solution prepared in step a) is added to a Sabouraud glucose medium to obtain an induction medium with a final concentration of 0.1-0.5 mg / mL; c) Hypha induction culture: Leucosporidium cryptocidum is inoculated into the induction medium, and is cultured at 28 DEG C for 24-48 hours to obtain filamentous Leucosporidium cryptocidum; d) Detection and confirmation: the hypha formation is observed under a microscope, and the observation is carried out after 48 hours; if the hypha phase formation rate is greater than or equal to 85%, the stable hypha phase is obtained by combining a PCR molecular biology method.

[0009] Further, the carbon quantum dots have a particle size of 2-8 nm, have fluorescence characteristics, have an excitation wavelength of 360 nm, and have an emission wavelength of 440 nm.

[0010] Further, the pH value of the induction medium is adjusted to 6.0-6.5.

[0011] Further, the preparation of the induction medium is subjected to high-pressure sterilization at 121 DEG C for 15-20 minutes.

[0012] Further, the PCR molecular biology method confirmation is to amplify the ITS region of Leucosporidium cryptocidum by using specific primers, and the amplification fragment is 600 bp.

[0013] The method for inducing hypha phase of Leucosporidium cryptocidum based on carbon quantum dots is applied to the preparation of an animal infection model for studying the pathogenic mechanism of Leucosporidium cryptocidum.

[0014] The method for inducing hypha phase of Leucosporidium cryptocidum based on carbon quantum dots is applied to the preparation of a standardized hypha phase induction model for providing an anti-yeast fungus drug.

[0015] The method for inducing hypha phase of Leucosporidium cryptocidum based on carbon quantum dots is applied to the study of morphological transformation mechanism of pathogenic yeasts in the process of invading a host in vivo.

[0016] The application has the following beneficial effects: 1. High induction efficiency: the method for inducing hypha phase of Leucosporidium cryptocidum based on carbon quantum dots significantly improves the hypha formation rate, and the hypha formation rate is increased from 40-60% in a traditional method to more than 85%.

[0017] 2. Good stability: the induction conditions of the present application are controllable, and the reproducibility is high, which provides stable materials for experimental research.

[0018] 3. Strong specificity: the present application combines morphological and molecular biological double confirmation to ensure the accuracy of mycelium identification.

[0019] 4. Simple operation: the present application uses hydrothermal method to prepare carbon quantum dots, one-step medium preparation, and mycelium induction culture conditions are easy to control, and the experimental process is simplified.

[0020] 5. Low cost: the present application uses cheap and easy-to-obtain raw materials to prepare carbon quantum dots, and the research cost is low.

[0021] 6. The present application helps to provide stable mycelium phase materials for pathogenic research of Candida macednioides, and has the following research application value: the present application can efficiently obtain mycelium phase materials, which is convenient for research on the expression of specific virulence factors of mycelium phase, and is beneficial to the research on pathogenic mechanism; stable induction conditions are helpful for the research on the molecular mechanism of yeast phase-mycelium phase transformation, which is beneficial to the research on morphological transformation mechanism; the present application provides a standardized mycelium phase induction model for the development of antifungal drugs, which can provide experimental materials for drug sensitivity test and invasion mechanism research of pathogenic yeast; the present application supports the comparative research on physiological and biochemical characteristics of the fungus in different morphologies, which is beneficial to the research on pathogenic biology. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The present application is a method for inducing Candida macednioides mycelium phase based on carbon quantum dots, and the steps are as follows: DETAILED DESCRIPTION

[0023] The present application will be described in detail below in combination with the drawings and examples.

[0024] Example: The present application is a method for inducing Candida macednioides mycelium phase based on carbon quantum dots, and the steps are as follows: a) Preparation and characterization of carbon quantum dots: Carbon quantum dots are prepared by hydrothermal method: 1. Take 2.0 g of citric acid and 1.0 g of urea, dissolve in 20 mL of deionized water, and stir until completely dissolved; 2. Transfer the solution to the inner liner of the hydrothermal reactor; 3. Put the hydrothermal reactor into a 230°C oven and react for 15 hours; 4. Naturally cool to room temperature to obtain a carbon quantum dot solution; 5. Adjust the concentration to 0.5 mg / mL; The carbon quantum dots obtained in step a) are added to a Sabouraud glucose medium to a final concentration of 0.5 mg / mL; the pH is adjusted to 6.0-6.5, and high-pressure sterilization is performed at 121 ℃ for 15 min to obtain an induction medium; c) Mycelium induction culture: The standard strain or clinical isolate of Starmerella cactiarum is inoculated into the Sabouraud glucose medium containing 0.5 mg / mL carbon quantum dots (i.e., the induction medium), and incubation is performed at 28 ℃ for 24-48 h to obtain the mycelial phase of Starmerella cactiarum; Morphological observation of the mycelial phase induced by carbon quantum dots: After 48 h of microscopic examination, a wet mount is prepared from the colony, and the formation of mycelium is observed under an optical microscope; The results show that: 1. Control group (without carbon quantum dots): mainly oval single-cell yeast morphology, with a small amount of pseudomycelium; 2. Experimental group (containing carbon quantum dots): a large amount of long and thin mycelial structure, continuous mycelium, and obvious branching, with a mycelial phase formation rate of ≥85%; d) Detection confirmation, using PCR molecular biology methods: DNA is extracted from the mycelial phase induced by carbon quantum dots and the control group, and PCR amplification is performed using ITS universal primers, and the primer sequences are as follows: Forward primer: 5'-TCCGTAGGTGAACCTGCGG-3'; Reverse primer: 5'-TCCTCCGCTTATTGATATGC-3'; The product is detected by 1.5% agarose gel electrophoresis. The results show that specific bands of about 600 bp are amplified in both groups, and the sequencing results confirm that the ITS sequence of Starmerella cactiarum is confirmed, and the mycelial phase induced by carbon quantum dots is Starmerella cactiarum.

[0025] The carbon quantum dots described in the application have a particle size of 2-8 nm, have fluorescence characteristics, an excitation wavelength of 360 nm, and an emission wavelength of 440 nm. Through the unique properties of carbon quantum dots, the induction efficiency and stability of the mycelial phase of Starmerella cactiarum are significantly improved, which provides a new technical means for in-depth study of the pathogenic mechanism of the pathogenic fungus, and has important scientific value and application prospects.

[0026] The method for inducing the mycelial phase of Starmerella cactiarum based on carbon quantum dots described in the application can be applied in the preparation of an animal infection model for studying the pathogenic mechanism of Starmerella cactiarum. Alternatively, it can be applied in the preparation of a standardized mycelial phase induction model for studying anti-yeast fungal drugs. In addition, it can be applied in the study of the morphological transformation mechanism of yeast pathogenic bacteria during invasion of the host body.

[0027] The parts not described in detail in the present application are all existing conventional technologies, and here, no further description is given.

[0028] It can be understood that the above specific description of the present application is only used to illustrate the present application and is not limited to the technical solutions described in the embodiments of the present application. Those skilled in the art should understand that the present application can still be modified or replaced equivalently to achieve the same technical effect; as long as the use needs are met, it is within the protection scope of the present application.

Claims

1. A method for inducing the mycelial phase of Leucomyces ramosus based on carbon quantum dots, characterized by, The method comprises the following steps: a) hydrothermal preparation of carbon quantum dots: 2.0 g of citric acid and 1.0 g of urea are dissolved in 20 mL of deionized water, and hydrothermal reaction is carried out at 230°C for 15 hours to obtain a carbon quantum dot solution; b) preparation of an induction medium: the carbon quantum dot solution prepared in step a) is added to a Sabouraud glucose medium to obtain an induction medium with a final concentration of 0.1-0.5 mg / mL; c) mycelium induction culture: the M. capitata is inoculated into the induction medium, and cultured at 28°C for 24-48 hours to obtain filamentous M. capitata; d) detection and confirmation: the mycelium formation is observed under a microscope, and the observation is carried out after 48 hours; if the mycelium phase formation rate is ≥ 85%, the stable mycelium phase is obtained by combining PCR molecular biology methods.

2. The method of claim 1, wherein: The carbon quantum dots have a particle size of 2-8 nm, have fluorescence characteristics, an excitation wavelength of 360 nm, and an emission wavelength of 440 nm.

3. The method of claim 1, wherein: The pH value of the induction medium is adjusted to 6.0-6.

5.

4. The method of claim 1, wherein: The preparation of the induction medium is subjected to high-pressure sterilization at 121°C for 15-20 minutes.

5. The method of claim 1, wherein: The PCR molecular biology method confirmation is the amplification of the ITS region of M. capitata by using specific primers, and the amplification fragment is 600 bp.

6. Use of the method according to any one of claims 1-5 in the preparation of an animal infection model for studying the pathogenic mechanism of M. capitata.

7. Use of the method according to any one of claims 1-5 in the preparation of a standardized mycelium phase induction model for studying anti-yeast fungal drugs.

8. Use of the method according to any one of claims 1-5 in the study of the morphological transformation mechanism of pathogenic yeast-like bacteria in the process of invading the host body.

Images