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Reagent capable of rapidly and quantitatively detecting sensitivity of antibacterial agent and application method of reagent

A technology for quantitative detection of antibacterial drugs, applied in the field of rapid detection of reagents for the sensitivity of pathogenic microorganisms to antibacterial drugs, can solve the problems of cumbersome operation, difficult to use in grass-roots hospitals, and small gradients of antibacterial drug concentrations, and achieve reduced operating steps and luminous intensity Stable, low-hardware-required effects

Inactive Publication Date: 2017-12-01
XINTRUM PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The Chinese patent "Rapid Drug Sensitivity Test Kit" (ZL201120469811) adopts the dilution method for drug sensitivity test, shortens the test time by the color detection method, but due to the low sensitivity of the color detection method, it takes 9-11 hours to get the drug Min results
[0009] Microplate-type luminescence detectors that are rarely equipped in primary medical institutions must be used, and an additional automatic sampling system is required
[0010] The second is that the operation is more cumbersome, so this technology is more difficult to use in primary hospitals:
[0015] The third is that it cannot meet the requirements of MIC (Minimum Inhibitory Concentration) determination:
[0016] The concentration gradient of antibacterial drugs set in this method is small, and MIC (minimum inhibitory concentration) cannot be determined
Therefore, this method only reports sensitivity qualitatively and does not provide exact MIC values

Method used

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  • Reagent capable of rapidly and quantitatively detecting sensitivity of antibacterial agent and application method of reagent
  • Reagent capable of rapidly and quantitatively detecting sensitivity of antibacterial agent and application method of reagent
  • Reagent capable of rapidly and quantitatively detecting sensitivity of antibacterial agent and application method of reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of bacterial ATP detection reagent

[0067] Follow the steps below to prepare bacterial ATP detection reagents:

[0068] (1) Use freshly prepared high-purity water to prepare 100mL 150-250mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution; add 0.15-1.20g chlorhexidine acetate, 1.5-2.5 mL Triton X-100 (Triton X-100), 0.3-0.5g magnesium chloride hexahydrate, 0.7-1.0g disodium ethylenediaminetetraacetic acid dihydrate (EDTA); use 1M sodium hydroxide to adjust the pH of the solution After 7.5, use a 0.22 μm filter membrane to filter and sterilize; add 20-50 mg luciferase (Sigma) and 100-200 mg D-luciferin (Sigma), dissolve, and store at -20°C.

[0069] Or (2) Use freshly prepared high-purity water to prepare 100mL 150-250mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) solution; add 0.06-0.10g cetyltrimethyl Ammonium bromide (CTAB), 0.15-1.20g chlorhexidine acetate, 1.5-2.5mL Triton X-100 (Triton X-100), 0.3-0.5g magnesium...

Embodiment 2

[0071] Example 2 Bacteria ATP detection reagent detects bacteria

[0072] In order to investigate the performance of the bacterial ATP detection reagent, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) were resuscitated on TSA plate medium and cultured at 37°C for 18-24 hours. Dilute the two bacteria with CAMHB broth to a series of concentrations, transfer a certain volume of the diluted bacteria solution to the blank multi-well plate, add a certain volume of the bacterial ATP detection reagent prepared in Example 1, and put it into the multi-function plate reader (PerkinElmer), detect the luminescence value of each well, repeat three wells for each concentration.

[0073] Table 1 and Table 2 show the luminescence values ​​of different amounts of Escherichia coli and Staphylococcus aureus, respectively. figure 1 with figure 2 Shown is the relationship between the detected luminous intensity and the number of bacteria. It can be seen from the figure that the l...

Embodiment 3

[0079] Example 3 Detecting the number of bacteria after antibiotic action

[0080] In order to detect the changes in the number of bacteria after 2 hours of antibiotic action, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 29213) were resuscitated on TSA plate medium and cultured at 37°C for 18-24 hours. In a 96-well plate, use column 1 as a negative control, and column 6 as a positive control; row A is cefazolin, rows B and E are piperacillin, rows C and G are netilmicin, and row D is levofloxacin , Line F is minocycline. Add 50μL of CAMHB broth to each well of column 1 to 6 of the well plate, and dilute the antibiotics with CAMHB broth to the required concentration (cefazolin 4μg / mL, piperacillin 16μg / mL, netilmicin 2μg / mL, levofloxacin 0.125μg / mL, minocycline 1μg / mL), add 50μL of the antibiotic diluent of the above concentration in column 2 to the corresponding row, and use the stepwise dilution method to dilute the antibiotic to 3-5 Remove the extra 50μL sol...

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Abstract

A kit capable of rapidly and quantitatively detecting sensitivity of an antibacterial agent is provided with a reagent capable of directly detecting bacteria ATP. After the reagent is added at a time, bacteria lysis and releasing and detection of ATP are realized at one step. By the kit, a drug sensitivity experimental result can be obtained within 2 hours. Requirements on hardware are low, any micropore plate type luminous detecting instrument can be used, and an automatic sample injection system is not required. The method is simple and convenient to operate, one-step sample adding is implemented, and lysate and an ATP detecting reagent are not required to be injected separately. An MIC value can be determined, and whether drug resistance exists or not can be reported simultaneously.

Description

Technical field: [0001] The invention relates to a reagent for rapidly detecting the sensitivity of pathogenic microorganisms to antibacterial drugs and an application method thereof. Background technique: [0002] The conventional Antimicrobial Susceptibility Tests (AST) methods currently used in clinical microbiology laboratories mainly include paper diffusion method, dilution method, and E-test method. In these methods, the results are judged by observing the growth of microorganisms by naked eyes, and it is often necessary to incubate for more than 16 hours before reporting the drug sensitivity results. [0003] The Chinese patent "Rapid Drug Sensitivity Test Kit" (ZL201120469811) uses the dilution method for drug susceptibility testing, which shortens the test time through the color detection method, but due to the low sensitivity of the color detection method, it takes 9-11 hours to get the drug Sensitive result. [0004] Although automated instruments have greatly improved t...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/76G01N33/569
CPCG01N21/6428G01N21/6452G01N21/763G01N33/56916G01N33/56938G01N33/9446G01N2333/245G01N2333/31
Inventor 羌维兵童明庆张怡
Owner XINTRUM PHARMA
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