A culture medium and fungal drug sensitivity test kit for improving Candida albicans tailing

Abstract

The present invention relates to C12Q1, and more specifically, to a culture medium and a fungal drug sensitivity test kit for improving the tailing of Candida albicans. The raw materials of the culture medium for improving the tailing of Candida albicans include a basal culture medium, a sulfonic acid buffer, a polyhydroxy aldehyde, a mineral substance and an inhibitor. The basal culture medium containing propyl glutathione and 4-hydroxyethylpiperazineethanesulfonic acid has excellent compatibility with mineral substances while providing a basic environment for fungal growth, so that nutrients are not easy to precipitate; the plant tissue culture antibacterial agent and quaternary ammonium salt with a weight ratio of (0.2-0.6): (0.03-0.1) can not only make propyl glutathione more stable, but also significantly improve the tailing phenomenon of in vitro drug sensitivity experiments. After reading the minimum inhibitory concentration value for 24h, there is basically no bacterial growth at 48h, and basically no interference is caused to the interpretation of MIC.

Description

Technical Field

[0001] The present invention relates to C12Q1, and more specifically, to a culture medium and a fungal drug sensitivity kit for improving the candida tailing condition. Background Art

[0002] The tailing phenomenon refers to the phenomenon that bacteria still grow in high-concentration drug solutions above the minimum inhibitory concentration (MIC).

[0003] Patent No. CN104833756B provides a method for determining the content of monoester alkaloids of glycosides, and optimizes the mobile phase and chromatographic column during the test to improve the tailing factor. "Study on the in vitro antifungal sensitivity and tailing phenomenon of Candida albicans to triazole drugs" (Chinese Journal of Leprosy and Skin Diseases) proposed that although the use of spectrophotometer method still cannot avoid the tailing phenomenon caused by drug dissolution, it can reduce the misjudgment of MIC value to a certain extent.

[0004] When conducting in vitro drug sensitivity tests on yeast samples, flucytosine and azole drugs are partially inhibited, which affects the interpretation (MIC) results of these drugs, and the tailing phenomenon is obvious, especially the tailing growth at 48 hours, which leads to a significant increase in MIC during interpretation. In vivo studies have confirmed that the increase in MIC caused by this tailing growth is actually still in the sensitive category. This phenomenon is mainly related to several points: first, the 24-hour growth cycle is not enough for Candida to grow to the maximum extent. In addition to the growth of high-concentration bacteria, the bacterial count also increases significantly at 48 hours; second, it is related to the antibacterial mechanism of azole drugs; third, it may be related to the upregulation of drug-resistant genes. At present, people often make some improvements to the detection methods, but such improvements cannot fundamentally alleviate the tailing phenomenon, and there are still large errors in the interpretation of MIC. Summary of the invention

[0005] In order to solve the above problems, the first aspect of the present invention provides a culture medium for improving the tailing of Candida albicans. The raw materials of the culture medium for improving the tailing of Candida albicans include basal culture medium, sulfonic acid buffer, polyhydroxy aldehyde, minerals and inhibitors.

[0006] Further preferably, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 33-36 g sulfonic acid buffer, 10-20 g basal culture medium, 15-25 g polyhydroxy aldehyde, 2.5-4 g minerals, and 0.03-0.08 g inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 45-50° C. and poured into a beaker or culture dish to obtain a culture medium for improving the tailing condition of Candida albicans.

[0007] Further preferably, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 34.53 g sulfonic acid buffer, 10 g basal culture medium, 20 g polyhydroxy aldehyde, 3.7 g minerals, and 0.045 g inhibitor are added to 1 L of double distilled water, heated to boiling, heating is stopped after boiling, cooled to 45-50° C., and poured into a beaker or culture dish to obtain a culture medium for improving the tailing condition of Candida albicans.

[0008] As a preferred technical solution of the present invention, the basic culture medium includes tripeptides and vitamin B.

[0009] As a preferred technical solution of the present invention, the tripeptide is selected from one or more of copper peptide, glutathione, collagen tripeptide, leupeptin, γ-L-glutamyl-L-α-aminobutyryl glycine, γ-L-glutamyl-L-alanyl glycine, prolyl-leucyl-glycine, ACVδ-(L-α-aminoadipyl)-L-Cys-D-Val.

[0010] Preferably, the tripeptide is glutathione. In the basal medium, the content of glutathione is 0.0005-0.002 g / L.

[0011] As a preferred technical solution of the present invention, the vitamin B is selected from one or more of vitamin B1, vitamin B2, vitamin B4, vitamin B5, and vitamin B12.

[0012] Preferably, vitamin B is vitamin B12. In the basal medium, the content of vitamin B12 is 0.000001-0.00001 g / L.

[0013] As a preferred technical solution of the present invention, the pH buffer range of the sulfonic acid buffer is 6-8.

[0014] Preferably, the pH buffer range of the sulfonic acid buffer is 6.3-7.9.

[0015] As a preferred technical solution of the present invention, the sulfonic acid buffer is selected from one or more of 2-(N-morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 3-(N-morpholino)-2-hydroxypropanesulfonic acid.

[0016] Preferably, the sulfonic acid buffer is 3-(N-morpholine)propanesulfonic acid.

[0017] As a preferred technical solution of the present invention, the mineral is selected from one or more of MnSO4·5H2O, CoCl2·6H2O, AlCl3·6H2O, CuCl2·H2O, ZnSO4·7H2O, FeSO4·7H2O, and Na2B4O4·10H2O.

[0018] Preferably, the minerals are MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O.

[0019] Further preferably, the weight ratio of MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O is (1-1.5):(0.7-1.2):(0.7-1.2):(0.7-1.2).

[0020] As a preferred technical solution of the present invention, the inhibitor is a plant tissue culture antibacterial agent and / or a quaternary ammonium salt.

[0021] As a preferred technical solution of the present invention, the inhibitor is a plant tissue culture antibacterial agent and a quaternary ammonium salt.

[0022] Preferably, in the inhibitor, the weight ratio of the plant tissue culture antibacterial agent and the quaternary ammonium salt is (0.2-0.6): (0.03-0.1).

[0023] Further preferably, the plant tissue culture antimicrobial agent is PAA and PPM, and in the plant tissue culture antimicrobial agent, the weight ratio of PAA to PPM is (0.2-0.4): (0.05-0.2).

[0024] More preferably, in the plant tissue culture antibacterial agent, the weight ratio of PAA to PPM is 0.3:0.1.

[0025] The propyl glutathione in the basal medium is unstable in the solution, and it is easy to produce ammonia so that harmful substances appear in the culture medium, thereby causing errors in the interpretation of MIC. In order to reduce the above-mentioned error, the inventor unexpectedly found in the experiment that adding a small amount of inhibitors can slightly reduce this error. After a large number of experimental explorations, the applicant found that adding a specific inhibitor and further controlling the ratio of the inhibitor can not only make the propyl glutathione more stable, but also significantly improve the tailing phenomenon of the in vitro drug sensitivity experiment. The MIC point of 48h is obvious and is no longer disturbed by the tailing. This improvement in tailing is particularly effective when the traditional tailings such as fluconazole, voriconazole, and itraconazole are more serious. The applicant speculates that the inhibitor and other substances in the system act on the one hand to prolong the time of the drug acting in the cell, and on the other hand to hinder the path of acquired resistance of the bacterium.

[0026] Preferably, the polyhydroxy aldehyde is glucose.

[0027] A second aspect of the present invention provides a fungal drug sensitivity test kit, which includes a culture medium that improves the Candida tailing condition.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The basic culture medium containing tripeptides and vitamin B12 not only provides a basic environment for fungal growth, but also has excellent compatibility with minerals, so that nutrients are not easy to precipitate; a specific proportion of 3-(N-morpholine) propanesulfonic acid is added to the culture medium to reduce the generation of free radicals in the basic culture medium, thereby improving the detection accuracy of MIC; the weight ratio of MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O is controlled to be (1-1.5): (0.7-1.2): (0.7-1.2): (0.7-1.2), further providing a good culture medium environment, making the fungi in the culture medium grow more uniformly, being conducive to the effect of drugs, and thus being conducive to the interpretation of MIC. The plant tissue culture antimicrobial agent and quaternary ammonium salt with a weight ratio of (0.2-0.6): (0.03-0.1) can not only make propylglutathione more stable, but also significantly improve the tailing phenomenon of the in vitro drug sensitivity test. After reading the 24h minimum inhibitory concentration value, there is basically no bacterial growth at 48h, and it basically does not interfere with the interpretation of MIC. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 To test the performance, fluconazole was added to Example 1 and cultured for 48 hours;

[0031] Figure 2 To test the performance, fluconazole was added to Example 2 and cultured for 48 hours;

[0032] Figure 3 To test the performance, fluconazole was added to Example 4 and cultured for 48 hours;

[0033] Figure 4 To test the performance, fluconazole was added to the culture medium of Example 5 and cultured for 48 hours. DETAILED DESCRIPTION

[0034] Example

[0035] The raw materials for preparing the composition in the embodiment are all commercially available, among which the basal culture medium is from Wenzhou Kangtai Biotechnology Co., Ltd., and its name is RPMI1640, the CAS number of 3-(N-morpholine)propanesulfonic acid is 1132-61-2, PAA is purchased from Zhongke Ruitai, the product number is PPA001, PPM is purchased from Biolab, the product number is SY0624, and the quaternary ammonium salt is purchased from Zhongan United Technology Co., Ltd., and its product name is silicone quaternary ammonium salt antibacterial agent.

[0036] Example 1

[0037] This example provides a culture medium for improving the tailing condition of Candida albicans. The raw materials of the culture medium for improving the tailing condition of Candida albicans include a basic culture medium, a sulfonic acid buffer, a polyhydroxy aldehyde, minerals and an inhibitor.

[0038] The preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 34.48 g of sulfonic acid buffer, 10 g of basic culture medium, 20 g of polyhydroxy aldehyde, 3.7 g of minerals, and 0.045 g of inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 45° C. and poured into a beaker to obtain the culture medium for improving the tailing condition of Candida albicans.

[0039] The basic culture medium includes a tripeptide and vitamin B. The tripeptide is glutathione. In the basic culture medium, the content of glutathione is 0.0013 g / L. Vitamin B is vitamin B12. In the basic culture medium, the content of vitamin B12 is 0.000007 g / L.

[0040] The pH buffer range of the sulfonic acid buffer is 6.5-7.9. The sulfonic acid buffer is 3-(N-morpholine)propanesulfonic acid.

[0041] The minerals are MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O. The weight ratio of MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O is 1.3:0.8:0.7:0.9.

[0042] The inhibitor is a plant tissue culture antibacterial agent and a quaternary ammonium salt. In the inhibitor, the weight ratio of the plant tissue culture antibacterial agent to the quaternary ammonium salt is 0.4:0.05.

[0043] The plant tissue culture antimicrobial agent is PAA and PPM. In the plant tissue culture antimicrobial agent, the weight ratio of PAA to PPM is 0.3:0.1.

[0044] The polyhydroxy aldehyde is glucose.

[0045] Example 2

[0046] This example provides a culture medium for improving the tailing condition of Candida albicans. Different from Example 1, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 33.9 g sulfonic acid buffer, 5 g basal culture medium, 18 g polyhydroxy aldehyde, 3.5 g minerals, and 0.035 g inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 50°C and poured into a beaker to obtain a culture medium for improving the tailing condition of Candida albicans.

[0047] Example 3

[0048] This example provides a culture medium for improving the tailing condition of Candida albicans. Different from Example 1, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 35 g sulfonic acid buffer, 7.5 g basal culture medium, 23 g polyhydroxy aldehyde, 3.7 g minerals, and 0.045 g inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 50°C and poured into a beaker to obtain a culture medium for improving the tailing condition of Candida albicans.

[0049] Example 4

[0050] This example provides a culture medium for improving the tailing condition of Candida albicans. Different from Example 1, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 35 g sulfonic acid buffer, 10 g basal culture medium, 20 g polyhydroxy aldehyde, 4.8 g minerals, and 0.055 g inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 45°C and poured into a beaker to obtain a culture medium for improving the tailing condition of Candida albicans.

[0051] The weight ratio of MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O is 1.5:1.1:1:1.2.

[0052] Example 5

[0053] This example provides a culture medium for improving the tailing condition of Candida albicans. Different from Example 1, the preparation method of the culture medium for improving the tailing condition of Candida albicans is as follows: 34.88 g of sulfonic acid buffer, 10 g of basic culture medium, 21 g of polyhydroxy aldehyde, 3.5 g of minerals, and 0.004 g of inhibitor are added to 1 L of double distilled water, heated to boil, and heating is stopped after boiling. The culture medium is cooled to 50° C. and poured into a beaker to obtain a culture medium for improving the tailing condition of Candida albicans.

[0054] The inhibitor is a plant tissue culture antimicrobial agent, which is PAA and PPM. In the plant tissue culture antimicrobial agent, the weight ratio of PAA to PPM is 0.3:0.1.

[0055] Performance Test:

[0056] 1. Tailing phenomenon test: Fluconazole (CAS No. 86386-73-4) was added to the culture medium for improving the tailing of Candida obtained in Examples 1, 2, 4, and 5, respectively, so that the concentration of fluconazole was 16 μg / mL, and a liquid culture medium was obtained. 100 μL of Candida suspension (from Wenzhou Kangtai Biotechnology Co., Ltd.) was inoculated into the liquid culture medium, and the culture medium was observed at 35°C for 48 hours. Figure 1-4 It can be seen that compared with Examples 4 and 5, the number of colonies in Examples 1 and 2 is less, and the tailing effect is significantly improved.

[0057] 2. Repeated stability test: Voriconazole (CAS No. 137234-62-9) was added to the culture medium for improving the tailing of Candida obtained in Examples 1-5, so that the concentration of voriconazole was 8 μg / mL, and a liquid culture medium was obtained. 100 μL of Candida suspension (from Wenzhou Kangtai Biotechnology Co., Ltd.) was inoculated into the liquid culture medium, and the MIC of voriconazole was determined. The parallel experiment was repeated five times, and the RSD was calculated. The results are shown in Table 1:

[0058] Table 1

[0059] Example Repeatability (%) 1 0.65 2 0.72 3 0.75 4 1.10 5 1.25

Claims

1. A culture medium for improving the tailing of Candida albicans, characterized in that: The raw materials of the culture medium for improving the Candida tailing condition include basal medium, sulfonic acid buffer, polyhydroxy aldehyde, minerals and inhibitors; The preparation method of the culture medium for improving the tailing of Candida albicans is as follows: 34.48 g of sulfonic acid buffer, 10 g of basic culture medium, 20 g of polyhydroxy aldehyde, 3.7 g of minerals, and 0.045 g of inhibitor are added to 1 L of double distilled water, heated to boil, and the heating is stopped after boiling, and the culture medium is cooled to 45° C. and poured into a beaker to obtain the culture medium for improving the tailing of Candida albicans; The basic culture medium includes a tripeptide and vitamin B; the tripeptide is glutathione; in the basic culture medium, the content of glutathione is 0.0013 g / L; the vitamin B is vitamin B12; in the basic culture medium, the content of vitamin B12 is 0.000007 g / L; The pH buffer range of the sulfonic acid buffer is 6.5-7.9; the sulfonic acid buffer is 3-(N-morpholine)propanesulfonic acid; The minerals are MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O; the weight ratio of MnSO4·5H2O, ZnSO4·7H2O, FeSO4·7H2O and Na2B4O4·10H2O is 1.3:0.8:0.7:0.9; The inhibitor is a plant tissue culture antibacterial agent and a quaternary ammonium salt; in the inhibitor, the weight ratio of the plant tissue culture antibacterial agent to the quaternary ammonium salt is 0.4:0.05; The plant tissue culture antibacterial agent is PAA and PPM, and in the plant tissue culture antibacterial agent, the weight ratio of PAA to PPM is 0.3:0.1; The polyhydroxy aldehyde is glucose.

2. A fungal drug sensitivity test kit, characterized in that: The invention comprises the culture medium for improving the Candida tailing condition as claimed in claim 1.

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