Fast screening method of anti-bacilus tuberculosis medicine

Abstract

The invention discloses a fast screening method of an anti-bacilus tuberculosis medicine. The method comprises the following steps: adding a PHBS solution containing a medicine to be screened and serum into a bacilus tuberculosis suspension; incubating the mixture in an incubator at 20-36 DEG C for 3-5 days, then adding a Nitrocefin solution and continuing the incubation for 20-40 minutes; and measuring the OD500 value, wherein when the OD500 value is less than 0.64, the medicine to be screened is the anti-bacilus tuberculosis medicine. The method is substantially based on an in-vitro screening model of bacilus tuberculosis cellular level with the best representativeness, and the detection result has strong representativeness; the method can test through an elisa plate, has the advantages of small sample dosage, short detection period and relatively sensitive detection result, and can realize high-flux screening; the screening period is short, the incubation time of the bacilus tuberculosis is shortened to about 4 days, and the screening cost is low; and meanwhile, the pollution is avoided, and the screening accuracy is high.

Description

technical field

[0001] The invention relates to the technical field of medicine, in particular to a rapid screening method for anti-tuberculosis mycobacterium drugs. Background technique

[0002] Tuberculosis is an infectious disease infected with Mycobacterium tuberculosis, and it is one of the diseases with the longest history and the most serious consequences among the diseases that endanger human life and health. Half of the people who are in contact with Mycobacterium tuberculosis will be infected, and people who have been infected with Mycobacterium tuberculosis will have the risk of becoming a tuberculosis patient throughout their life, and 10% of people infected with Mycobacterium tuberculosis will develop the disease. Tuberculosis was once widespread all over the world. Since 1945, a variety of anti-tuberculosis chemical drugs came out, and the epidemic was once eased. However, due to stagnation in the development of anti-tuberculosis drugs and the increase in the ...

Examples

Embodiment 1

[0020] Example 1: Rapid screening of compounds PW-1 (tetrandrine), PW-2 (tetrandrine), PW-3 (betulinic acid), PW-4 (β-amyresinol) and compound PW- 5 (Ruikuling) whether it has anti-mycobacterium tuberculosis activity.

[0021] The compound was dissolved in PHBS solution to prepare a 2 mg / mL PHBS solution, which was autoclaved for later use. The Mycobacterium tuberculosis H37Rv strain was formulated into 2 × 10 8 CFU / mL of bacterial suspension, add 100 μL of bacterial suspension, 100 μL of compound solution and 100 μL of serum to a 96-well plate, and the concentration of the compound solution is 666 μg / mL. After culturing in a 36°C incubator for 4 days, add 100 μL of 5 mmol / l Nitrocefin solution to each well, then place in a 36°C incubator for 30 minutes, and then measure the OD with a microplate reader 500 value. Isoniazid was used as a positive control, and the wells without any drug were used as negative controls.

[0022] Results: OD of negative control (i.e. no drug a...

Embodiment 2

[0023] Example 2. Screening compounds PW-1 (tetrandrine), PW-2 (tetrandrine), PW-3 (betulinic acid), PW-4 (β-amyresin) and Whether the compound PW-5 (Ruikuling) has anti-mycobacterium tuberculosis activity.

[0024] 1. Sample configuration: compound PW-1, PW-2, PW-3, PW-4, PW-5 and positive control drug (isoniazid) with 50% DMSO aqueous solution to make 10mg / mL mother solution, use Store in a refrigerator at 4°C after sterilizing with a bacterial filter.

[0025] 2. Anti-tuberculosis test

[0026] Prepare medium: measure 150mL distilled water and add 0.7g KH 2 PO 4 , 0.06g MgSO4 , 0.15g magnesium citrate, 0.9g asparagine, 2.0g monosodium glutamate, heat to dissolve at 80°C, and add 7.5g potato flour when cooling to 60°C. Add 250mL of whole egg liquid and 5mL of 2% malachite green aqueous solution, and mix well. Add 3 mL of glycerin, 1.0 mL of Tween-80, 1.0 mL of 0.1% PHA, 10 mL of calf serum, 0.5 mL of 1% zinc sulfate, and 1 mL of niacin solution of 200 μg / mL after filter...

technical field

[0001] The invention relates to the technical field of medicine, in particular to a rapid screening method for anti-tuberculosis mycobacterium drugs. Background technique

[0002] Tuberculosis is an infectious disease infected with Mycobacterium tuberculosis, and it is one of the diseases with the longest history and the most serious consequences among the diseases that endanger human life and health. Half of the people who are in contact with Mycobacterium tuberculosis will be infected, and people who have been infected with Mycobacterium tuberculosis will have the risk of becoming a tuberculosis patient throughout their life, and 10% of people infected with Mycobacterium tuberculosis will develop the disease. Tuberculosis was once widespread all over the world. Since 1945, a variety of anti-tuberculosis chemical drugs came out, and the epidemic was once eased. However, due to stagnation in the development of anti-tuberculosis drugs and the increase in the ...

Examples

Embodiment 1

[0020] Example 1: Rapid screening of compounds PW-1 (tetrandrine), PW-2 (tetrandrine), PW-3 (betulinic acid), PW-4 (β-amyresinol) and compound PW- 5 (Ruikuling) whether it has anti-mycobacterium tuberculosis activity.

[0021] The compound was dissolved in PHBS solution to prepare a 2 mg / mL PHBS solution, which was autoclaved for later use. The Mycobacterium tuberculosis H37Rv strain was formulated into 2 × 10 8 CFU / mL of bacterial suspension, add 100 μL of bacterial suspension, 100 μL of compound solution and 100 μL of serum to a 96-well plate, and the concentration of the compound solution is 666 μg / mL. After culturing in a 36°C incubator for 4 days, add 100 μL of 5 mmol / l Nitrocefin solution to each well, then place in a 36°C incubator for 30 minutes, and then measure the OD with a microplate reader 500 value. Isoniazid was used as a positive control, and the wells without any drug were used as negative controls.

[0022] Results: OD of negative control (i.e. no drug a...

Embodiment 2

[0023] Example 2. Screening compounds PW-1 (tetrandrine), PW-2 (tetrandrine), PW-3 (betulinic acid), PW-4 (β-amyresin) and Whether the compound PW-5 (Ruikuling) has anti-mycobacterium tuberculosis activity.

[0024] 1. Sample configuration: compound PW-1, PW-2, PW-3, PW-4, PW-5 and positive control drug (isoniazid) with 50% DMSO aqueous solution to make 10mg / mL mother solution, use Store in a refrigerator at 4°C after sterilizing with a bacterial filter.

[0025] 2. Anti-tuberculosis test

[0026] Prepare medium: measure 150mL distilled water and add 0.7g KH 2 PO 4 , 0.06g MgSO4 , 0.15g magnesium citrate, 0.9g asparagine, 2.0g monosodium glutamate, heat to dissolve at 80°C, and add 7.5g potato flour when cooling to 60°C. Add 250mL of whole egg liquid and 5mL of 2% malachite green aqueous solution, and mix well. Add 3 mL of glycerin, 1.0 mL of Tween-80, 1.0 mL of 0.1% PHA, 10 mL of calf serum, 0.5 mL of 1% zinc sulfate, and 1 mL of niacin solution of 200 μg / mL after filter...