Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker

A detection method, amino acid technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc., can solve problems such as high equipment cost, high technical threshold, and wrong judgment.

Inactive Publication Date: 2020-07-28
RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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Problems solved by technology

For example, most microfluidic-based bacterial microscopic imaging technologies can directly observe the growth, proliferation or morphological changes of bacteria under the action of antibiotics. The amount of samples and reagents is greatly reduced, but microfluidic technology has not yet been popularized. The technical threshold for judging by microscopic imaging is high, the promotion is difficult, and the equipment cost is high. Moreover, based on the detection mechanism of bacterial growth, proliferation or morphological changes, it is impossible to identify the Bacteria in non-growing but metabolically active (NGMA) state, prone to misinterpretation [Refs 3, 4, 5]
The RNA detection method judges the sensitivity of bacteria to drugs by detecting the changes in the transcripts of bacteria under the action of antibiotics. Can determine whether the bacteria are sensitive to antibiotics, but cannot quantify the MIC [References 6, 7]
Although the atomic force microscope cantilever method judges the metabolic activity of bacteria by microscopic observation and monitoring the movem

Method used

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  • Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker
  • Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker
  • Antibacterial drug sensitivity test detection method based on fluorescent D-type amino acid metabolism marker

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Experimental program
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Embodiment 1

[0028] Detection of oxacillin (OX), vancomycin (VA), and erythromycin (E) against Gram-positive bacteria Staphylococcus aureus (S.aureus) with high levels of OX-resistant strain ST5, Effects of low-level OX-resistant strain ST59 and OX-sensitive strain ST398.

[0029] as attached image 3 The flow chart of the rapid detection method for antimicrobial susceptibility testing based on FDAA metabolic markers is shown, and the specific experimental steps are as follows:

[0030] 1. Pick the colony to be tested that was cultured on the plate overnight and mix it well in normal saline to make a uniform bacterial solution, dilute it into cation-adjusted broth medium (CAMHB) to make a bacterial solution with a concentration of OD600=0.2.

[0031] 2. Add 45 μl of different concentrations of the antibiotic solution to be tested to the 96-well plate (see Table 1 for the antibiotic concentration gradient setting).

[0032] 3. Add 50 μl of the bacterial solution prepared in step 1 to the ...

Embodiment 2

[0047] Using FDAA metabolic labeling method to detect the effects of cefepime (FEP), imipenem (IPM), levofloxacin (LEV) and tigecycline (TGC) on Gram-negative bacteria Escherichia coli (E. coli) drug-resistant strain 1113 and sensitive strain 1146.

[0048] The specific experimental steps are as follows:

[0049] 1. Pick the colony to be tested that was cultured on the plate overnight and mix well in normal saline to make a uniform bacterial solution, dilute it into cation-adjusted broth medium (CAMHB) to make a bacterial solution with a concentration of OD600=0.2.

[0050] 2. Add 45 μl of different concentrations of antibiotic solutions to be tested to the 96-well plate (see Table 3 for antibiotic concentration gradient settings).

[0051] 3. Add 50 μl of the bacterial solution prepared in step 1 to the 96-well plate, mix well, and incubate at 37°C in the dark for 2 hours.

[0052] 4. Add 5 μl of 10 mM Cy5-DAA probe (final concentration 0.5 mM, see attached image 3 ), aft...

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Abstract

The invention relates to an antibacterial drug sensitivity test detection method based on a fluorescent D-type amino acid metabolism marker. According to the method, the fluorescent D-type amino acidmetabolism marker is utilized to mark a bacterial solution obtained by mixing and incubating with an antibiotic solution, so that the minimum inhibitory concentration of antibiotics can be obtained according to the fluorescence signal intensity of the bacterial solution.

Description

technical field [0001] The present invention relates to the evaluation method of bacterial metabolic activity and the detection method of antimicrobial susceptibility test, especially to the detection method of antimicrobial susceptibility test based on fluorescent D-type amino acid metabolism label, for rapid evaluation or evaluation of antibacterial agent or bacterial metabolic activity, for example Antibiotics, antibacterial effect on clinically isolated bacterial strains, to guide clinical medication. Background technique [0002] With the widespread use of antibiotics in medical and farming industries, especially the extensive use of broad-spectrum antibiotics, the problem of bacterial resistance has become increasingly prominent, and even super bacteria that can resist almost all antibiotics have emerged one after another. The emergence and spread of bacterial resistance has attracted worldwide attention, and is considered by the World Health Organization to be one of ...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12Q1/18
CPCG01N21/64G01N21/6428C12Q1/18G01N2021/6417G01N2021/6439G01N33/582G01N15/1459G01N2015/0065C12Q1/02
Inventor 王炜杨朝勇
Owner RENJI HOSPITAL AFFILIATED TO SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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