阅读说明：本技术 一种中药单体药敏检测方法 (Method for detecting drug sensitivity of traditional Chinese medicine monomer ) 是由 邢海洋 瞿玲娜 于 2019-08-26 设计创作，主要内容包括：本发明公开一种中药单体药敏检测方法,采用微量肉汤二倍稀释法测定中药单体作用于细菌(包含多重耐药菌)的最低抑菌浓度,包括以下步骤：(1)将中药单体溶解于二甲基亚砜,配置成浓度为10N mg/ml的中药单体溶液；(2)取96孔板,从第二列起每孔加入二甲基亚砜10μl,第一列加入中药单体溶液20μl；(3)依次进行倍比稀释；(4)保存于温箱至二甲基亚砜全部挥发,得不同剂量梯度的中药单体；(5)将浊度为0.5的菌悬液加入96孔板,每孔100μl；37℃培养18h后观察结果；(6)结果判定：药物组出现无浑浊的最小浓度为中药单体的最低抑菌浓度(MIC)。本发明检测过程简单能够简便有效测试中药单体的药敏性,可用来指导临床中药治疗细菌感染性疾病。(The invention discloses a method for detecting drug sensitivity of a traditional Chinese medicine monomer, which adopts a trace broth two-fold dilution method to determine the minimum inhibitory concentration of the traditional Chinese medicine monomer acting on bacteria (including multiple drug-resistant bacteria), and comprises the following steps: (1) dissolving the traditional Chinese medicine monomer in dimethyl sulfoxide to prepare a traditional Chinese medicine monomer solution with the concentration of 10N mg/ml; (2) taking a 96-well plate, adding 10 mu l of dimethyl sulfoxide into each hole from the second row, and adding 20 mu l of traditional Chinese medicine monomer solution into the first row; (3) sequentially carrying out multiple dilution; (4) storing in a warm box until the dimethyl sulfoxide is completely volatilized to obtain the Chinese medicinal monomers with different dose gradients; (5) adding bacterial suspension with turbidity of 0.5 into a 96-well plate, wherein each well is 100 mu l; observing the result after culturing at 37 ℃ for 18 h; (6) and (4) judging a result: the minimum concentration of the drug group without turbidity is the Minimum Inhibitory Concentration (MIC) of the traditional Chinese medicine monomer. The invention has simple detection process, can simply, conveniently and effectively test the drug sensitivity of the traditional Chinese medicine monomer, and can be used for guiding the clinical traditional Chinese medicine to treat bacterial infectious diseases.)

1. A method for detecting drug sensitivity of a traditional Chinese medicine monomer is characterized by comprising the following steps: the method for determining the minimum inhibitory concentration of the traditional Chinese medicine monomer acting on bacteria (containing multiple drug-resistant bacteria) by adopting a trace broth two-fold dilution method comprises the following steps:

(1) Firstly, dissolving a traditional Chinese medicine monomer in dimethyl sulfoxide to prepare a traditional Chinese medicine monomer solution with the concentration of 10N mg/ml;

(2) Taking a 96-well plate, adding 10 mu l of dimethyl sulfoxide into each hole from the second row, and adding 20 mu l of the traditional Chinese medicine monomer solution prepared in the step (1) into the first row;

(3) Taking 10 mul of the cells in the first row of cell culture holes, adding the cells into the second row of cell culture holes, and sequentially carrying out multiple dilution;

(4) diluting in multiple proportion, storing in an incubator until dimethyl sulfoxide is completely volatilized, and remaining traditional Chinese medicine monomers with different dosage gradients in cell culture holes;

(5) adding bacterial suspension with turbidity of 0.5 into a 96-well plate, wherein each well is 100 mu l; the final concentration of the Chinese medicinal monomers is N, N/2, N/4, N/8, N/16, N/32, N/64, N/128, N/256 mg/mL; observing the result after culturing at 37 ℃ for 18 h;

(6) And (4) judging a result: the minimum concentration of the drug group without turbidity is the Minimum Inhibitory Concentration (MIC) of the traditional Chinese medicine monomer.

2. the method for detecting single drug sensitivity of traditional Chinese medicine according to claim 1, which is characterized in that: the Chinese medicinal monomer is baicalin.

3. the method for detecting single drug sensitivity of traditional Chinese medicine according to claim 1, which is characterized in that: the steps 1-6 are carried out under aseptic conditions.

4. the method for detecting single drug sensitivity of traditional Chinese medicine according to claim 3, which is characterized in that: the testing step is performed in a biosafety cabinet.

5. The method for detecting single drug sensitivity of traditional Chinese medicine according to claim 1, which is characterized in that: the 96-hole cell culture plate can simultaneously detect the drug sensitivity of a plurality of traditional Chinese medicine monomers at one time.

Technical Field

The invention relates to the technical field of drug sensitivity test, in particular to a method for detecting drug sensitivity of a traditional Chinese medicine monomer.

Background

At present, the phenomenon of antibiotic abuse clinically plays a certain diffusion role in the epidemic of multiple drug-resistant bacteria. The increasingly prominent problem of multiple drug-resistant bacteria presents a serious challenge to clinical anti-infective therapy.

The drug sensitivity experiment provides important reference value for the treatment of bacterial infectious diseases, especially the treatment of multiple drug-resistant bacterial infection. However, the existing drug sensitivity experiments are all in vitro bacteriostasis experiments of western antibacterial drugs.

research reports in recent years show that some traditional Chinese medicine components have inhibitory effects on various bacteria, especially multiple drug-resistant bacteria. But the clinical application still lacks the commercialized traditional Chinese medicine allergy experimental equipment which is simple, convenient and feasible in clinical application and low in cost.

At present, the traditional treatment based on syndrome differentiation is mainly used for treating bacterial infectious diseases in the traditional Chinese medicine, an accurate treatment means is lacked, and the clinical curative effect is poor. In the research of ' golden bridge engineering ' subject ' baicalin in vitro bacteriostatic experiment research on multiple drug-resistant bacteria by scientific cooperation in Wenling City in 2019, the applicant of the invention discovers a clinically feasible and low-cost Chinese medicinal monomer bacteriostatic experiment scheme, which can provide important reference value for guiding clinical Chinese medicinal antibacterium, especially for treatment of multiple drug-resistant bacteria.

Disclosure of Invention

in order to solve the existing problems, the invention provides a method for detecting the drug sensitivity of a traditional Chinese medicine monomer. The invention is realized by the following technical scheme.

A method for detecting drug sensitivity of a traditional Chinese medicine monomer is used for measuring the minimum inhibitory concentration of the traditional Chinese medicine monomer acting on bacteria (containing multiple drug-resistant bacteria) by adopting a trace broth two-fold dilution method, and comprises the following steps:

(1) firstly, dissolving a traditional Chinese medicine monomer in dimethyl sulfoxide to prepare a traditional Chinese medicine monomer solution with the concentration of 10N mg/ml;

(2) taking a 96-well plate, adding 10 mu l of dimethyl sulfoxide into each hole from the second row, and adding 20 mu l of the traditional Chinese medicine monomer solution prepared in the step (1) into the first row;

(3) Taking 10 mul of the cells in the first row of cell culture holes, adding the cells into the second row of cell culture holes, and sequentially carrying out multiple dilution;

(4) Diluting in multiple proportion, storing in an incubator until dimethyl sulfoxide is completely volatilized, and remaining traditional Chinese medicine monomers with different dosage gradients in cell culture holes;

(5) Adding bacterial suspension with turbidity of 0.5 into a 96-well plate, wherein each well is 100 mu l; the final concentration of the Chinese medicinal monomers is N, N/2, N/4, N/8, N/16, N/32, N/64, N/128, N/256 mg/mL; observing the result after culturing at 37 ℃ for 18 h;

(6) And (4) judging a result: the minimum concentration of the drug group without turbidity is the Minimum Inhibitory Concentration (MIC) of the traditional Chinese medicine monomer.

preferably, the traditional Chinese medicine monomer is baicalin.

preferably, the steps 1-6 are performed under aseptic conditions.

Preferably, the testing step is performed in a biosafety cabinet.

Preferably, the 96-well cell culture plate can simultaneously carry out drug sensitivity detection on a plurality of traditional Chinese medicine monomers at one time.

The invention has the beneficial effects that:

The method for detecting the drug sensitivity of the traditional Chinese medicine monomer is simple in detection process, a common 96-well plate is used as a practical tool, the drug sensitivity of the traditional Chinese medicine monomer can be simply, conveniently and effectively tested, clinical traditional Chinese medicine can be guided to treat bacterial infectious diseases, especially multiple drug-resistant bacteria infectious diseases, and the aim of accurately treating the traditional Chinese medicine is fulfilled.

Detailed Description

The technical solution of the present invention will be described in more detail and fully with reference to the following embodiments.