Method for accelerating color development speed of filamentous fungus resazurin and application of method

Abstract

The invention discloses a method for accelerating the color development speed of filamentous fungus resazurin and application of the method, and relates to the technical field of drug sensitivity detection. The invention provides a method for accelerating the color development speed of filamentous fungus resazurin, which comprises the following steps: S1, diluting a filamentous fungus bacterial liquid by using an MUELLER-HINTON culture medium, and adding the diluted filamentous fungus bacterial liquid into a to-be-detected drug hole for incubation; s2, performing suction filtration after incubation; s3, adding a color developing agent containing a catalyst for color development; s4, result interpretation; the catalyst in the step S3 is menadione. According to the MUELLER-HINTON culture medium, in the Aspergillus drug sensitivity detection process based on resazurin color development, after a catalyst is added, color development is accelerated, meanwhile, all medicine adding holes cannot be red, and the MIC value can be accurately obtained.

Description

Technical Field

[0001] This invention relates to the field of drug sensitivity testing technology, specifically to a method for accelerating the color development speed of filamentous fungi, *Rhododendron simsii*, and its application. Background Technology

[0002] Antifungal susceptibility testing is a test to detect the sensitivity of fungi to different antifungal drugs. It is used to guide clinicians in selecting appropriate antifungal drugs for treatment, improving treatment efficacy, and reducing adverse drug reactions and the development of drug resistance.

[0003] Traditional methods for drug susceptibility testing mainly include the disk diffusion method, the E test, and the broth dilution method. The disk diffusion method involves placing a disk containing a certain amount of drug on a culture medium coated with yeast suspension, and observing the diameter of the inhibition zone to determine drug sensitivity. This method is simple to operate, but it does not provide a direct MIC value, and the results are affected by various factors. The E test combines the principles of the dilution and diffusion methods, using a test strip with an exponentially decreasing concentration of antimicrobial drug to directly quantify the minimum inhibitory concentration (MIC) of the test drug against the test bacteria. However, this test strip is expensive, and determining the MIC endpoint for some drugs can be challenging. The broth dilution method can be used to quantitatively test the in vitro activity of antimicrobial drugs against specific bacteria and fungi. The dilution method is accurate and has a wide range of applications, but it is cumbersome, time-consuming, and labor-intensive, making it less suitable for clinical use.

[0004] The common drawback of the aforementioned conventional methods is that drug susceptibility testing requires 24-72 hours to complete, which is insufficient to meet the urgent clinical need for rapid drug administration. Because filamentous fungi require even longer culture times, drug susceptibility testing takes at least 48-72 hours, leading to the death of many critically ill patients due to delayed treatment. Therefore, there is an urgent need to improve the speed of drug susceptibility testing for filamentous fungi.

[0005] Currently, in the Aspergillus susceptibility testing process based on rezinol color development, while adding a catalyst can accelerate the color development, it also causes all wells to turn red, making it impossible to obtain the MIC value. Therefore, there is a need for a method that can accelerate the color development speed of Aspergillus rezinol while still obtaining the MIC value. Summary of the Invention

[0006] The purpose of this invention is to provide a method for accelerating the color development speed of filamentous fungi, specifically *Zygodium japonicum*, and its application.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] On one hand, the present invention provides a method for accelerating the color development speed of filamentous fungi azurite, comprising the following steps:

[0009] S1. The filamentous fungal culture was diluted with MUELLER-HINTON medium and then added to the wells containing the test reagents for incubation.

[0010] S2. After incubation, perform vacuum filtration;

[0011] S3. Add a colorimetric reagent containing a catalyst to develop the color;

[0012] S4. Result Interpretation;

[0013] The catalyst mentioned in step S3 is menaquinone.

[0014] Specifically, the filamentous fungi mentioned in step S1 include, but are not limited to, one or more of the following genera: Aspergillus, Mucorales, Penicillium, Pythium, Entomycetes, Basilaria, Penicillium, Porphyromonas, Fusarium, Cladosporium, Cladosporium, Colletosporium, Cetacea, Cetacea, Cetacea, Beauveria bassiana, Metarhizium anisopliae, Trichoderma, Schizophyllum, Terminalia, Trichoderma, Thermophila, Coprinus, Trichophyton, Epidermophyton, Microsporum, Trichoderma, Cladosporium, Cladosporium, Chaetomium, Cladosporium, Chlorophytum, Chlorophytum, Alternaria, Hypnosporium, Hypnosporium, Curvularia, Helicobacter, Cladosporium ...

[0015] Furthermore, the Aspergillus species mentioned include, but are not limited to: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidus, Aspergillus variegata, Aspergillus polymorpha, Aspergillus terreus, Aspergillus pyrophorus, Aspergillus spp., Aspergillus spp., Aspergillus lanceolata, Aspergillus oryzae, Aspergillus scabra, Aspergillus oryzae, Aspergillus oryzae, Aspergillus scabra, Aspergillus ochreus.

[0016] Furthermore, the solutions used to prepare the bacterial solution in step S1 include, but are not limited to, sterile physiological saline, PBS, and / or sterile water.

[0017] Preferably, the solution used to prepare the bacterial solution in step S1 is sterile water.

[0018] Specifically, the McFarland turbidity unit of the bacterial solution described in step S1 is 0.5-1.5.

[0019] More specifically, the McFarland turbidity unit of the bacterial solution described in step S1 is 0.5-1.0 or 1.0-1.5.

[0020] Preferably, the McFarland turbidity unit of the bacterial solution in step S1 is 0.5-1.0.

[0021] More preferably, the McFarland turbidity unit of the bacterial solution in step S1 is 0.5 or 1.0.

[0022] Specifically, in step S1, the mold culture was diluted to a concentration of 0.3 × 10⁻⁶ using MUELLER-HINTON medium. 5 CFU / mL - 1×10 6 CFU / mL.

[0023] Further, in step S1, the mold culture was diluted with MUELLER-HINTON medium to a concentration of (0.3-2)×10⁻⁶. 5 CFU / mL.

[0024] Specifically, the culture media selected for incubation in step S2 include, but are not limited to: DMEM medium, RPMI 1640 medium, Mueller-Hinton broth, SDB medium, PDB medium, YM medium, MEM medium, LB medium, SOB medium, SOC medium, TB medium, 2×YT medium and / or YPD medium; preferably, the culture medium is Mueller-Hinton broth.

[0025] Specifically, the incubation described in step S2 includes incubation at 35°C for 30-120 minutes.

[0026] Furthermore, the final concentration of the colorimetric catalyst in step S3 is 0.1-5 mM; preferably, the concentration of the colorimetric catalyst in step S3 is 0.1-2 mM, and even more preferably, the concentration of the colorimetric catalyst in step S3 is 0.24 mM.

[0027] More preferably, the filtration method in step S2 is as follows: transfer to a microporous filter plate for filtration, or the drug sensitivity card can be directly filtered.

[0028] Specifically, the colorimetric agents mentioned in step S3 include, but are not limited to: resamarazine solution, WST, methylene blue solution, neutral red solution, and / or methyl violet solution.

[0029] Preferably, the color developer in step S3 is a razor blue solution.

[0030] Furthermore, the concentration of the resamarium solution is 5-30 μg / ml;

[0031] Furthermore, the concentration of the razor cyan solution is 18 μg / ml.

[0032] In another aspect, the present invention provides the application of the combination of MUELLER-HINTON medium and menadione in accelerating the detection speed of drug susceptibility in filamentous fungi.

[0033] Specifically, the filamentous fungal culture was diluted using MUELLER-HINTON medium;

[0034] Furthermore, the filamentous fungal culture was diluted to a concentration of 0.3 × 10⁻⁶ using MUELLER-HINTON medium. 5 CFU / mL - 1×10 6 CFU / mL.

[0035] Furthermore, the filamentous fungal culture was diluted using MUELLER-HINTON medium to a concentration of (0.3-2)×10⁻⁶. 5 CFU / mL.

[0036] Specifically, the concentration of the bacterial solution can be (0.3-2)×10⁻⁶. 5 CFU / mL, specifically 0.3×10 5 CFU / mL, 0.4×10 5 CFU / mL, 0.5×10 5 CFU / mL, 0.6×10 5 CFU / mL, 0.7×10 5 CFU / mL, 0.8×10 5 CFU / mL, 0.9×10 5 CFU / mL, 1×10 5 CFU / mL, 1.1×10 5 CFU / mL, 1.2×10 5 CFU / mL, 1.3×10 5 CFU / mL, 1.4×10 5 CFU / mL, 1.5×10 5 CFU / mL, 1.6×10 5 CFU / mL, 1.7×10 5 CFU / mL, 1.8×10 5 CFU / mL, 1.9×10 5 CFU / mL, 2×10 5 CFU / mL, etc., and other point values ​​within this range can be selected.

[0037] Specifically, the filamentous fungi include, but are not limited to, one or more of the following genera: Aspergillus, Mucorales, Penicillium, Pythium, Entomophthora, Basilaria, Penicillium, Porphyromonas, Fusarium, Cladosporium, Cladosporium, Colletosporium, Cetacea, Cetacea, Cetacea, Cladosporium, Beauveria bassiana, Metarhizium anisopliae, Trichoderma, Schizophyllum, Terminalia, Trichoderma, Thermophila, Coprinus, Trichophyton, Epidermophyton, Microsporum, Trichoderma, Cladosporium, Cladosporium, Chaetomium, Cladosporium, Chlorophytum, Chlorophytum, Alternaria, Hypnosporium, Hypnosporium, Curvularia, Helicobacter, Cladosporium ...

[0038] Furthermore, the Aspergillus species mentioned include, but are not limited to: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidus, Aspergillus variegata, Aspergillus polymorpha, Aspergillus terreus, Aspergillus pyrophorus, Aspergillus spp., Aspergillus spp., Aspergillus lanceolata, Aspergillus oryzae, Aspergillus scabra, Aspergillus oryzae, Aspergillus oryzae, Aspergillus scabra, Aspergillus ochreus.

[0039] Specifically, the drug in question is an antifungal drug.

[0040] Furthermore, the antifungal drugs mentioned include, but are not limited to: polyene drugs, allylamine drugs, pyrrole drugs, nucleoside drugs, and echinocandins.

[0041] Specifically, the polyene drugs include, but are not limited to: amphotericin B, amphotericin B lipid complex, amphotericin B cholesterol sulfate, amphotericin B liposomes, nystatin aureus, clotrimazole B, tebuconazole, phlebotomycin, hanmycin, nystatin, epimycin, pimomycin and / or trachomycin.

[0042] Specifically, the allylamine drugs include, but are not limited to: naftifine, terbinafine, and / or butenafine.

[0043] Specifically, the pyrrole drugs include, but are not limited to, imidazole drugs and / or triazole drugs.

[0044] Furthermore, the imidazole drugs include, but are not limited to: ketoconazole, miconazole, econazole, sertaconazole and / or clotrimazole.

[0045] Furthermore, the triazole drugs mentioned include, but are not limited to: itraconazole, fluconazole, voriconazole, isaconazole, posaconazole, and rivconazole.

[0046] Specifically, the echinocandins include, but are not limited to: caspofungin, micafungin, rezafenamicin, eurifenamicin, or anidoxurin.

[0047] In another aspect, the present invention provides the application of the above-described method in accelerating the detection speed of drug susceptibility in filamentous fungi.

[0048] The beneficial effects of this invention are as follows:

[0049] The MUELLER-HINTON medium of the present invention can accelerate the color development in the Aspergillus susceptibility detection process based on resporane color development by adding a catalyst, without causing all the wells to turn red, and can still obtain the MIC value. Attached Figure Description

[0050] Figure 1 The results of the amphotericin B susceptibility test to Aspergillus fumigatus in Example 1 are shown.

[0051] Figure 2 The results of the amphotericin B susceptibility test to aflatoxin B in Example 2 are shown.

[0052] Figure 3 The results of the amphotericin B susceptibility test to Aspergillus niger in Example 3 are shown.

[0053] Figure 4 The results of the amphotericin B susceptibility test to Aspergillus nidulans in Example 4 are shown.

[0054] Figure 5 The results of the amphotericin B susceptibility test to aflatoxin in Comparative Example 1 are shown.

[0055] Figure 6 The results of the amphotericin B susceptibility test to aflatoxin in Comparative Example 2 are shown. Detailed Implementation

[0056] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0057] Example 1: Rapid drug susceptibility testing of amphotericin B against Aspergillus fumigatus

[0058] 1. Preparation of Aspergillus fumigatus solution: Aspergillus fumigatus is prepared with sterile water to form an Aspergillus fumigatus solution with an MCF of 0.5.

[0059] 2. Adding samples

[0060] (1) Set up wells of different concentrations of test drugs (the test drug solution is serially diluted) and positive control wells (PC wells) in 96-well deep well plates or drug sensitivity cards, and add 450 μL of amphotericin B solution with concentrations of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL and 0.25 μg / mL and MUELLER-HINTON medium, respectively.

[0061] (2) Count the Aspergillus fumigatus in the solution and dilute with MUELLER-HINTON broth to 2×10⁻⁶. 5 The concentration of CFU / mL was used to obtain the diluted bacterial solution. 450 μL of the diluted bacterial solution was added to each well.

[0062] 3. Incubation: Incubate at 35℃ for 2 hours and then remove.

[0063] 4. Vacuum filtration: Transfer to a microporous filter plate for vacuum filtration; drug sensitivity cards can be directly vacuum filtered.

[0064] 5. Add a colorimetric reagent containing a catalyst: the final concentration of the resamarium solution is 18 μg / ml, and the final concentration of menaquinone is 2 mM. Observe the color change at 35℃.

[0065] Result interpretation rules: Observe whether the positive control well turns pink. If it does, interpret the result and read the minimum drug concentration at which the color remains completely unchanged (remaining blue). Figure 1 As shown, the time for color development was 4 hours.

[0066] Example 2: Rapid drug susceptibility testing of amphotericin B against Aspergillus flavus

[0067] 1. Preparation of Aspergillus flavus solution: Aspergillus flavus is prepared with sterile water to form an Aspergillus flavus solution with an MCF of 0.5.

[0068] 2. Adding samples

[0069] (1) Set up test drug wells and positive control wells (PC wells) in 96-well deep well plates or drug sensitivity cards, and add 450 μL of amphotericin B solution with concentrations of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL and MUELLER-HINTON medium, respectively.

[0070] (2) The Aspergillus flavus culture was counted and diluted with MUELLER-HINTON broth to a concentration of 0.3 × 10⁻⁶. 5 The concentration of CFU / mL was used to obtain the diluted bacterial solution. 450 μL of the diluted bacterial solution was added to each well.

[0071] 3. Incubation: Incubate at 35℃ for 2 hours and then remove.

[0072] 4. Vacuum filtration: Transfer to a microporous filter plate for vacuum filtration; drug sensitivity cards can be directly vacuum filtered.

[0073] 5. Add a colorimetric reagent containing a catalyst: the final concentration of the resamarine solution is 18 μg / ml, and the final concentration of menaquinone is 0.24 mM. Observe the color change at 35℃.

[0074] Experimental results: The results are as follows Figure 2 As shown, the time for color development was 4 hours.

[0075] Example 3: Rapid drug susceptibility testing of amphotericin B against Aspergillus niger.

[0076] 1. Preparation of Aspergillus inoculum: Aspergillus niger is prepared into an Aspergillus inoculum with an MCF of 0.5 using sterile water.

[0077] 2. Adding samples

[0078] (1) Set up test drug wells and positive control wells (PC wells) in 96-well deep well plates or drug sensitivity cards, and add 450 μL of amphotericin B solution with concentrations of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL and MUELLER-HINTON medium, respectively.

[0079] (2) Count the Aspergillus niger suspension and dilute it to 1×10⁻⁶ with MUELLER-HINTON broth. 5 The concentration of CFU / mL was used to obtain the diluted bacterial solution. 450 μL of the diluted bacterial solution was added to each well.

[0080] 3. Incubation: Incubate at 35℃ for 2 hours and then remove.

[0081] 4. Vacuum filtration: Transfer to a microporous filter plate for vacuum filtration; drug sensitivity cards can be directly vacuum filtered.

[0082] 5. Add a colorimetric reagent containing a catalyst: the final concentration of the resamarium solution is 18 μg / ml, and the final concentration of menaquinone is 0.1 mM. Observe the color change at 35℃.

[0083] Experimental results: such as Figure 3 As shown, the time for color development was 4 hours.

[0084] Example 4: Rapid drug susceptibility testing of amphotericin B against Aspergillus nidulans.

[0085] 1. Preparation of Aspergillus nidus culture: Aspergillus nidus culture is prepared with sterile water to form an Aspergillus culture culture with an MCF of 0.5.

[0086] 2. Adding samples

[0087] (1) Set up test drug wells and positive control wells (PC wells) in 96-well deep well plates or drug sensitivity cards, and add 450 μL of amphotericin B solution with concentrations of 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL and MUELLER-HINTON medium, respectively.

[0088] (2) Count the Aspergillus nidulans suspension and dilute with MUELLER-HINTON broth to 1×10⁻⁶. 5 The concentration of CFU / mL was used to obtain the diluted bacterial solution. 450 μL of the diluted bacterial solution was added to each well.

[0089] 3. Incubation: Incubate at 35℃ for 2 hours and then remove.

[0090] 4. Vacuum filtration: Transfer to a microporous filter plate for vacuum filtration; drug sensitivity cards can be directly vacuum filtered.

[0091] 5. Add a colorimetric reagent containing a catalyst: the final concentration of the resamarine solution is 18 μg / ml, and the final concentration of menaquinone is 5 mM. Observe the color change at 35℃.

[0092] Experimental results: such as Figure 4 As shown, the time for color development was 4 hours.

[0093] Comparative Example 1

[0094] The only difference from Example 2 is that step 2, which involves adding samples, is different. Instead, the Aspergillus flavus solution is diluted using 1640 medium. Specifically, (2) the Aspergillus flavus solution is counted and diluted with 1640 medium to 0.3 × 10⁻⁶. 5 The concentration of CFU / mL was used to obtain the diluted bacterial solution. 450 μL of the diluted bacterial solution was added to each well, and the rest were the same.

[0095] Experimental results: such as Figure 5 As shown, the color development time was 4 hours, but all the dosing wells turned red with no color gradient, making it impossible to interpret the MIC.

[0096] Comparative Example 2

[0097] The only difference from Example 2 is that no catalyst was added in step 5. Instead, a colorimetric agent was added: the final concentration of the resamarium solution was 18 μg / ml, and the color change was observed at 35°C. All other steps were the same.

[0098] Experimental results: The results are as follows Figure 6 As shown, all wells were blue after 4 hours of development.

[0099] Comparative Example 3

[0100] The only difference from Example 2 is that the concentration of the catalyst is different in step 5. Specifically, a colorimetric agent containing the catalyst is added: the final concentration of the resamarine solution is 18 μg / ml, and the final concentration of menaquinone is 6 mM. The color change is observed at 35°C.

[0101] Experimental results: The color development time was observed to be 4 hours, but all the dosing wells turned red, with no color gradient, making it impossible to determine the MIC.

[0102] Comparative Example 4

[0103] The only difference from Example 2 is that the type of catalyst is different in step 5. Specifically, a colorimetric agent containing the catalyst is added: the final concentration of the resamarine solution is 18 μg / ml, the final concentration of 1,4-naphthoquinone is 0.24 mM, and the color change is observed at 35°C.

[0104] Experimental results: After 4 hours of observation, all holes were blue.

Claims

1. A method for accelerating the color development speed of filamentous fungi in azurite, characterized in that, Includes the following steps: S1. The filamentous fungal culture was diluted with MUELLER-HINTON medium and then added to the wells containing the test reagents for incubation. S2. After incubation, perform vacuum filtration; S3. Add a colorimetric reagent containing a catalyst to develop the color; S4. Result Interpretation; The catalyst mentioned in step S3 is menaquinone.

2. The method according to claim 1, characterized in that, The filamentous fungi mentioned include one or more of the following genera: Aspergillus, Mucorales, Penicillium, Pythium, Entomycetes, Basilaria, Penicillium, Porphyromonas, Fusarium, Cladosporium, Cladosporium, Colletosporium, Cetacea, Cetacea, Cetacea, Cladosporium, Beauveria bassiana, Metarhizium anisopliae, Trichoderma, Schizophyllum, Terminalia, Trichoderma, Thermophila, Coprinus, Trichophyton, Epidermophyton, Microsporum, Trichoderma, Cladosporium, Cladosporium, Chaetomium, Cladosporium, Chlorophytum, Cladosporium ...

3. The method according to claim 1, characterized in that, The catalyst mentioned in step S3 is menadione, and the final concentration of menadione is 0.1-5 mM.

4. The method according to claim 1, characterized in that, In step S1, the mold culture was diluted to a concentration of 0.3 × 10⁻⁶ using MUELLER-HINTON medium. 5 CFU / mL - 1×10 6 CFU / mL.

5. The method according to claim 1, characterized in that, The color-developing agent mentioned in step S3 is a resazurin solution; the concentration of the resazurin solution is 5-30 μg / ml.

6. Application of the combination of MUELLER-HINTON medium and menadione in accelerating the detection speed of drug susceptibility in filamentous fungi.

7. The application according to claim 6, characterized in that, To dilute the filamentous fungal culture to a concentration of 0.3 × 10⁻⁶ using MUELLER-HINTON medium. 5 CFU / mL - 1×10 6 CFU / mL.

8. The application according to claim 7, characterized in that, To dilute the filamentous fungal culture to a concentration of (0.3-2)×10⁻⁶ using MUELLER-HINTON medium. 5 CFU / mL.

9. The application according to claim 6, characterized in that, The drug in question is an antifungal agent.

10. The application according to claim 9, characterized in that, The antifungal drugs mentioned include polyene drugs, allylamine drugs, pyrrole drugs, nucleoside drugs, and echinocandins.

11. The application of the method according to any one of claims 1-5 in accelerating the detection speed of drug susceptibility testing for filamentous fungi.

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