High-throughput drug sensitivity detection kit as well as use method and application thereof

A technology for detection kits and detection reagents, applied in the direction of microorganism-based methods, biochemical equipment and methods, specific-purpose bioreactors/fermenters, etc. It is economical, easy to use, easy to use, and highly clinically relevant.

Pending Publication Date: 2020-08-07
HANGZHOU HANJING BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, molecular biology methods have many steps, and the detection cost of each drug is high.
Colorimetric detection is a multi-step operation, carried out in a microplate, and the accuracy is not high
It can be seen that there are some defects in the existing drug sensitivity detection methods of tuberculosis.

Method used

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  • High-throughput drug sensitivity detection kit as well as use method and application thereof
  • High-throughput drug sensitivity detection kit as well as use method and application thereof
  • High-throughput drug sensitivity detection kit as well as use method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] In this experimental example, Mycobacterium tuberculosis was cultured in a single test tube by EZMTT method and turbidity method respectively.

[0071] EZMTT method: Mix live bacteria, detection reagents and Middle brook 7H9 medium at a volume ratio of 1:0.1:20, add to a 4ml plastic single tube, store and culture at 37°C, measure its absorbance at OD450nm every day, And draw the growth curve. The detection reagent used in this experiment contained 0.5 mM tetrazolium monosulfonate and 50 μM phenazine methyl sulfate.

[0072] Turbidimetric method: Mix live bacteria with Middle brook 7H9 medium at a volume ratio of 1:20, add to 4ml plastic single tube, store and culture at 37°C, measure its absorbance at OD610nm every day, and draw the growth curve .

[0073] In this experiment, the living bacteria were selected as Mycobacterium tuberculosis, and their concentrations were selected as A) 1.25×10 4 CFU / mL, B) 6.25×10 3 CFU / mL, C) 3.13×10 3 CFU / mL, D) 1.56×10 3 CFU / mL, ...

Embodiment 2

[0078] This example provides a high-throughput drug susceptibility detection kit using a microwell plate as a culture container. Due to the large difference in the culture environment between a single test tube and a microwell plate, there are many uncertain factors. Therefore, the present invention compares micropore plate and single test tube culture, and has determined the doubling time and Z factor of EZMTT method to cultivate viable bacteria in micropore plate.

[0079] In this experiment, EZMTT method (OD450nm) and turbidity method (OD610nm) were used to culture Mycobacterium tuberculosis in 4ml plastic single tube and 96-well plate respectively, and its growth curve was measured ( Figure 5 ), wherein the inoculation concentration of Mycobacterium tuberculosis is 1.25×10 5 CFU / mL. From Figure 5 As can be seen in the figure, the use of EZMTT method to cultivate Mycobacterium tuberculosis in 4ml plastic single tube (Tube) and 96-well plate (Plate) is not as sensitive a...

Embodiment 3

[0082] This embodiment provides a high-throughput drug susceptibility detection kit, including a culture container, a culture medium and a detection reagent.

[0083] The culture container used in this embodiment is: 96-well plate;

[0084] The medium used in this example is: Middle brook 7H9 medium;

[0085] The detection reagent used in this embodiment contains 0.5 mM tetrazolium monosulfonate and 50 μM phenazine methyl sulfate;

[0086] The live bacterial strain used in the present embodiment is: M.tuberculosis H37Ra, and the concentration of the live bacteria is: 2×10 5 CFU / ml;

[0087] The drugs to be tested selected in this embodiment are: isoniazid (INH), rifampicin (RIF), ethambutol (EMB), pyrazinamide (PZA), streptomycin sulfate (STR), amida Cardine (AMK), levofloxacin (LVX), moxifloxacin hydrochloride (MVX), prothionamide (PTO), linezolid (LZO), bedaquiline (BEDA).

[0088] Preferably, the final concentration (ug / ml) of the above-mentioned 11 drugs to be tested i...

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Abstract

The invention belongs to the technical field of drug screening, and provides a high-throughput drug sensitivity detection kit as well as a use method and application thereof. The high-throughput drugsensitivity detection kit comprises a culture container, a culture medium and a detection reagent, and the detection reagent is a mixture of phenazine methyl sulfate derivatives and an oxidation reagent containing tetrazole salt. The high-throughput drug sensitivity detection kit provided by the invention has the characteristics of rapidness, accuracy, economy, simplicity, convenience, safety, notoxicity, visual inspection and high clinical correlation, is simple in use method, has higher accuracy and sensitivity, and can screen a large number of antibacterial drugs at one time.

Description

technical field [0001] The invention relates to the technical field of drug sensitivity detection, in particular to a high-throughput drug sensitivity detection kit and its use method and application. Background technique [0002] Tuberculosis (Tuberculosis, TB) is a chronic airborne disease caused by Mycobacterium tuberculosis. Although Mycobacterium tuberculosis mainly infects the lungs, it can almost invade various organs of the human body. It has always been a threat Major public health issues of global human health. Nontuberculosis mycobacteria (NTM) refers to all mycobacteria except Mycobacterium tuberculosis and Bacillus leprae, also known as environmental mycobacteria. Non-tuberculous mycobacteria widely exist in soil, dust, water, fish and poultry in nature, and the main route of transmission is to acquire infection from the environment, such as sewage, while human-to-human infection is extremely rare. Non-tuberculous mycobacteria are opportunistic pathogens. Cert...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/18C12Q1/04C12M1/34C12R1/32
CPCC12Q1/18C12Q1/04
Inventor 阮奔放
Owner HANGZHOU HANJING BIOTECHNOLOGY CO LTD
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