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Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method

A technique for detecting Mycobacterium tuberculosis and its detection method, which is applied in the detection of Mycobacterium tuberculosis drug susceptibility phenotype and its application field, and can solve the problems of the unreported application of the drug susceptibility detection method of Mycobacterium tuberculosis, the threat to the safety of drug sensitivity testing personnel, Drug susceptibility testing personnel are prone to infection and other problems, achieving the effect of short detection time, small workload and high accuracy

Inactive Publication Date: 2011-08-10
LANZHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this drug susceptibility testing method has a serious disadvantage, that is, the biological safety is poor, and the drug susceptibility testing personnel are susceptible to infection.
During the culture process of Mycobacterium tuberculosis in 96-well plate, tuberculosis aerosol will be produced, drug sensitivity testing personnel need to open the 96-well plate during the process of adding the indicator resazurin, tuberculosis aerosol will be safe for drug sensitivity testing personnel pose a serious threat to laboratory transmission of tuberculosis
After reviewing the literature, there is no report on the drug susceptibility detection method of Mycobacterium tuberculosis and its application in the screening of anti-tuberculosis drugs that can make up for the above defects

Method used

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  • Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method
  • Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method
  • Mycobacterium tuberculosis medicament sensitive phenotype detection method and application of method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] 1. Preparation of 0.2% redox indicator resazurin

[0032] Accurately weigh 20 mg of the indicator resazurin with an electronic balance and put it into a beaker, then fully dissolve it with 10 ml of analytically pure methanol, and store it in a 4°C refrigerator for later use.

[0033] 2. Preparation of centrifuge tubes with redox indicator resazurin

[0034] Such as figure 1 As shown, take several 0.5ml centrifuge tubes (Eppendorf Company), open the tube cover of the centrifuge tube, make the centrifuge tube cover vertical to the centrifuge tube, insert the centrifuge tube into the centrifuge tube rack, and then upward Add 4 ul of the prepared 0.2% resazurin methanol solution, and after the methanol is completely evaporated at room temperature, as figure 2 , put into an autoclavable aluminum or plastic box, autoclave at 121°C for 20 minutes, and take it out for later use.

[0035] 3. Drug susceptibility testing of Mycobacterium tuberculosis

[0036] Inoculate 30 mg ...

Embodiment 2

[0040] 1. Preparation of 0.2% redox indicator resazurin

[0041] Accurately weigh 20 mg of the indicator resazurin with an electronic balance and put it into a beaker, then fully dissolve it with 10 ml of analytically pure methanol, and store it in a 4°C refrigerator for later use.

[0042] 2. Preparation of centrifuge tubes with redox indicator resazurin

[0043] Take several 1.0ml centrifuge tubes (Eppendorf Company), open the cap of the centrifuge tube, make the cap of the centrifuge tube perpendicular to the centrifuge tube, insert the centrifuge tube into the centrifuge tube rack, and then add the prepared 2.5ul of 0.2% resazurin methanol solution, after fully volatilizing the methanol at room temperature, put it into an autoclavable aluminum box or plastic box, sterilize with damp heat and autoclave at 121°C for 20 minutes, and take it out for use.

[0044] 3. Drug susceptibility testing of Mycobacterium tuberculosis

[0045] Inoculate 5 mg of the three kinds of Mycoba...

Embodiment 3

[0049] 1. Preparation of 0.2% redox indicator resazurin

[0050] Accurately weigh 20 mg of the indicator resazurin with an electronic balance and put it into a beaker, then fully dissolve it with 10 ml of analytically pure methanol, and store it in a 4°C refrigerator for later use.

[0051] 2. Preparation of centrifuge tubes with redox indicator resazurin

[0052] Take several 1.5ml centrifuge tubes (Eppendorf Company), open the cap of the centrifuge tube, make the cap of the centrifuge tube perpendicular to the centrifuge tube, insert the centrifuge tube into the centrifuge tube rack, and then add the prepared 6ul of 0.2% resazurin methanol solution, let the methanol completely volatilize at room temperature, put it into an autoclavable aluminum box or plastic box, sterilize with damp heat and high pressure at 121°C for 20 minutes, and take it out for use.

[0053] 3. Drug susceptibility testing of Mycobacterium tuberculosis

[0054] Take 50 mg of the three kinds of Mycobac...

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Abstract

The invention discloses a mycobacterium tuberculosis medicament sensitive phenotype detection method, which comprises the following steps of: 1) adding 2.5 to 6mul of 0.2 percent of resazurin dissolved by using methanol with mass percentage concentration into the inner side of a tube cover of a sterile centrifuge tube, and sterilizing the centrifuge tube after the methanol is volatilized; 2) inoculating 5 to 50mg of mycobacterium tuberculosis to be detected into a 7H9-S culture medium, regulating the concentration of the bacteria solution by adopting turbidimetry, regulating the concentration of the bacteria solution to the concentration of a Mcburney turbidimetric 1 tube by using sterile water or 0.9-percent physiological saline, and diluting the bacteria solution by 20 times by using the 7H9-S culture medium; 3) inoculating 50 to 150mu of diluted bacteria solution into the centrifuge tube of the step 1); 4) adding 100mul of anti-tubercular medicament solution diluted by 7H9-S and to be detected into the centrifuge tube of the step 3), wherein the concentration of the medicament to be detected is 0.01mug / ml to 2.5mg / ml; 5) after the centrifuge tube of the step 4) is covered closely, culturing the solution for 6 to 7 days at the temperature of 37 DEG C; and 6) inverting and oscillating the centrifuge tube of the step 5), and culturing the solution for 12 to 48 hours at the temperature of 37 DEG C after the solution becomes blue. Proved by experiments, in the mycobacterium tuberculosis medicament sensitive phenotype detection method and application of the mycobacterium tuberculosis medicament sensitive phenotype detection method in anti-tubercular medicament screening, the method has low workload and short detection time; compared with a traditional L-J medium medicament sensitive detection method, the accuracy reaches 90 to 99 percent; and the method has good biological safety, does not cause laboratory propagation of tuberculosis, is suitable for large-scale.

Description

technical field [0001] The invention relates to a method for detecting drug-sensitive phenotype and its application, in particular to a method for detecting drug-sensitive phenotype of Mycobacterium tuberculosis and its application in screening anti-tuberculosis drugs. Background technique [0002] Tuberculosis is a highly contagious disease worldwide, causing more than one million deaths each year. In recent years, with the emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistant Mycobacterium tuberculosis (XDR-TB), the treatment of tuberculosis has become a great challenge (Espinal M A, Laszlo A , Simonsen L, et al. N Engl J Med. 2001, 344:1294-1303). Due to the emergence of MDR-TB and XDR-TB, tuberculosis drug susceptibility testing and drug resistance testing are particularly important, which can not only better manage and treat tuberculosis patients, reduce the spread of drug-resistant bacteria, but also greatly reduce new tub...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/04
Inventor 汪月祝秉东田丽丽张颖高娃
Owner LANZHOU UNIVERSITY
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